Non-human animals having a humanized lymphocyte-activation gene 3

ABSTRACT

Non-human animals, and methods and compositions for making and using the same, are provided, wherein the non-human animals comprise a humanization of a Lymphocyte activation gene 3 (Lag3). The non-human animals may be described, in some embodiments, as having a genetic modification to an endogenous Lag3 locus so that the non-human animals express a Lag3 polypeptide that includes a human portion and an endogenous portion (e.g., a non-human portion).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/258,181, filed Nov. 20, 2015, and U.S. Provisional Application No.62/370,430, filed Aug. 3, 2016, the entire contents of which areincorporated herein by reference.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The Sequence Listing in the ASCII text file, named as34031_10212US01_SequenceListing.txt of 49 KB, created on Oct. 18, 2016,and submitted to the United States Patent and Trademark Office viaEFS-Web, is incorporated herein by reference.

BACKGROUND

Cancer remains a tremendous challenge in the healthcare industryworldwide, in part, because cancer cells possess the ability to evadethe host immune system. Such ability has been understood to be theresult of inhibition and/or down-regulation of anti-tumor immunity.Still, development of useful in vivo systems to optimally determine thetherapeutic potential of new cancer therapeutics and/or therapeuticregimens that are designed to activate and/or promote anti-tumorimmunity and determine the mechanisms of how cancer cells provideinhibitory signals to immune cells, in particular, T cells, is lacking.Such systems provide a source for assays for assessing the therapeuticefficacy of candidate agents that promote an anti-tumor environment invivo.

SUMMARY

The present invention encompasses the recognition that it is desirableto engineer non-human animals to permit improved systems for identifyingand developing new therapeutics and/or therapeutic regimens that can beused for the treatment of cancer. The present invention also encompassesthe recognition that it is desirable to engineer non-human animals topermit improved in vivo systems for identifying and developing newtherapeutics that can be used to treat autoimmune (or inflammatory, orinfectious) diseases, disorders or conditions. The present inventionalso encompasses the recognition that it is desirable to engineernon-human animals to permit improved in vivo systems for identifying anddeveloping new therapeutics that promote anti-tumor immunity. Further,the present invention also encompasses the recognition that non-humananimals having a humanized Lymphocyte-activation gene 3 (Lag-3) and/orotherwise expressing, containing, or producing a human or humanizedLag-3 polypeptide are desirable, for example for use in identifying anddeveloping cancer therapeutics that up-regulate anti-tumor immunity. Insome embodiments, non-human animals of the present invention provideimproved in vivo systems for the identification and development ofcombination therapies that include targeting Lag-3 and/or Programmedcell death 1 (PD-1).

In some embodiments, the present invention provides non-human animalshaving a genome comprising an engineered Lag-3 gene, which engineeredLag-3 gene includes genetic material from two different species (e.g., ahuman and a non-human). In some embodiments, such an engineered Lag-3gene includes genetic material that encodes one or moreimmunoglobulin-like (Ig-like) domains of a human LAG-3 polypeptide. Insome embodiments, genetic material encodes the Ig-like domains of ahuman LAG-3 polypeptide that are responsible for ligand binding (e.g.,MHC class II binding). Thus, in some embodiments, an engineered Lag-3gene of a non-human animal as described herein encodes a Lag-3polypeptide that contains human and non-human portions, wherein thehuman and non-human portions are linked together and form a functionalLag-3 polypeptide. In some embodiments, an engineered Lag-3 gene of anon-human animal as described herein encodes a Lag-3 polypeptide thatcontains the first two Ig-like domains (D1 and D2), in whole or in part,of a human LAG-3 polypeptide. Generally speaking, the first two Ig-likedomains (D1 and D2) are contained within the N-terminal 260 amino acidsof a human LAG-3 polypeptide; and in some embodiments, within amino acidresidues 21-260, 23-260 or 29-260 of a human LAG-3 polypeptide. See,also, FIGS. 1-2, for example.

In some embodiments, a non-human animal is provided, that expresses aLag-3 polypeptide, which Lag-3 polypeptide comprises a human portion andan endogenous portion.

In some embodiments, a non-human animal is provided, whose genomecomprises a humanized Lag-3 gene (or locus) that comprises an endogenousportion and a human portion, wherein the endogenous and human portionsare operably linked to a non-human Lag-3 promoter.

In some embodiments, an endogenous portion of a Lag-3 polypeptidecomprises an intracellular portion of an endogenous Lag-3 polypeptide.In some embodiments, the endogenous portion of a Lag-3 polypeptidecomprises the intracellular domain of an endogenous Lag-3 polypeptide.In some certain embodiments, an endogenous portion of Lag-3 polypeptidefurther comprises a transmembrane portion of an endogenous Lag-3polypeptide. In some embodiments, the endogenous portion of a Lag-3polypeptide comprises the transmembrane domain of an endogenous Lag-3polypeptide. In some embodiments, the endogenous portion of a Lag-3polypeptide further comprises an extracellular portion of an endogenousLag-3 polypeptide, e.g., a C-terminal portion of the extracellulardomain of an endogenous Lag-3 polypeptide that does not include thefirst two Ig-like domains but includes the last two Ig-like domains (D3and D4). In some embodiments, the endogenous portion of a Lag-3polypeptide comprises amino acids of the signal peptide of an endogenousLag-3 polypeptide. For example, the endogenous portion of a Lag-3polypeptide comprises substantially the signal peptide of an endogenousLag-3 polypeptide.

In some embodiments, an endogenous portion of Lag-3 polypeptide has anamino acid sequence that is at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% identical to a correspondingamino acid sequence that appears in a rodent Lag-3 polypeptide of SEQ IDNO:2 or SEQ ID NO:4. In some embodiments, an endogenous portion of Lag-3polypeptide has an amino acid sequence that is substantially identicalor identical to a corresponding amino acid sequence that appears in arodent Lag-3 polypeptide of SEQ ID NO:2 or SEQ ID NO:4.

In some embodiments, an endogenous portion of a humanized Lag-3 genecomprises endogenous non-human Lag-3 exons 1, 5, 6, 7 and 8. In someembodiments, exons 1, 5, 6, 7 and 8 of an endogenous non-human Lag-3gene are at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 98% identical to the corresponding exons 1, 5, 6,7 and 8 that appear in a rodent Lag-3 mRNA sequence of SEQ ID NO:1 orSEQ ID NO:3. In some embodiments, exons 1, 5, 6, 7 and 8 of anendogenous non-human Lag-3 gene are substantially identical or identicalto the corresponding exons 1, 5, 6, 7 and 8 that appear in a rodentLag-3 mRNA sequence of SEQ ID NO:1 or SEQ ID NO:3.

In some embodiments, a human portion (or human sequence) of a humanizedLag-3 polypeptide comprises one or more of the Ig-like domains of ahuman LAG-3 polypeptide that are responsible for ligand binding (e.g.,MHC class II binding). In some embodiments, the human portion of ahumanized Lag-3 polypeptide comprises the first two Ig-like domains (D1and D2), in whole or in part, of a human LAG-3 polypeptide. In someembodiments, within amino acid residues 21-260, 23-260 or 29-260 of ahuman LAG-3 polypeptide. In some embodiments, the human portion of ahumanized Lag-3 polypeptide comprises amino acids 29-260 (or 23-260 or21-260) of a human LAG-3 polypeptide. In some embodiments, a humanportion (or human sequence) of a Lag-3 polypeptide comprises an aminoacid sequence that is at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 98% identical to a corresponding aminoacid sequence that appears in a human LAG-3 polypeptide of SEQ ID NO:6.In some embodiments, a human portion (or human sequence) of a Lag-3polypeptide comprises an amino acid sequence that is substantiallyidentical or identical to a corresponding amino acid sequence thatappears in a human LAG-3 polypeptide of SEQ ID NO:6.

In some embodiments, a human portion (or human sequence) of a humanizedLag-3 gene encodes at least amino acids 29-260 (or 23-260 or 21-260) ofa human LAG-3 polypeptide. In some embodiments, a human portion (orhuman sequence) of a humanized Lag-3 gene comprises exons 2-4 (or exons2, 3 and 4) of a human LAG-3 gene.

In some embodiments, exons 2-4 of a human LAG-3 gene are least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98%identical to the corresponding exons 2-4 that appear in a human LAG-3mRNA sequence of SEQ ID NO:5. In some embodiments, exons 2-4 of a humanLAG-3 gene are substantially identical or identical to the correspondingexons 2-4 that appear in a human LAG-3 mRNA sequence of SEQ ID NO:5. Insome embodiments, a human portion of a humanized Lag-3 gene comprises asequence that is codon-optimized for expression in a non-human animal asdescribed herein.

In some embodiments, a Lag-3 polypeptide that comprises a human portionand an endogenous portion is encoded by a nucleic acid sequence placedat an endogenous Lag-3 locus (or gene) as described herein.

In some embodiments, an endogenous portion of Lag-3 polypeptide isencoded by endogenous Lag-3 exons 1, 5, 6, 7 and 8.

In some embodiments, a humanized Lag-3 gene includes endogenousnon-human Lag-3 exon 1, human LAG-3 exons 2-4, and non-human Lag-3 exons5-8, wherein the non-human and human exons are operably linked to eachother and to an endogenous non-human Lag-3 promoter. In someembodiments, such humanized Lag-3 gene is placed at an endogenous Lag-3locus.

In some embodiments, a humanized Lag-3 gene encodes a humanized Lag-3polypeptide that includes a signal peptide that is substantiallyidentical to the signal peptide of an endogenous non-human Lag-3polypeptide; an extracellular domain that includes a human portion and anon-human portion wherein the human portion comprises the first twoIg-like domains (e.g., a sequence comprising amino acids 26-290) of ahuman LAG-3 polypeptide and the non-human portion comprises the last twoIg-like domains of an endogenous non-human Lag-3 polypeptide; and thetransmembrane and intracellular domains of an endogenous non-human Lag-3polypeptide.

In some embodiments, a Lag-3 polypeptide produced or expressed by anon-human animal as described herein is provided. In some embodiments, aLag-3 polypeptide produced or expressed by a non-human animal asdescribed herein is translated in a cell of the non-human animal with anon-human signal peptide, in whole or in part (e.g., a chimeric signalpeptide). In some embodiments, a Lag-3 polypeptide produced or expressedby a non-human animal as described herein comprises an amino acidsequence that is at least 50%, at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 98% identical to an amino acid sequence thatappears in a humanized Lag-3 polypeptide of SEQ ID NO:8. In someembodiments, a Lag-3 polypeptide produced or expressed by a non-humananimal as described herein comprises an amino acid sequence that issubstantially identical or identical to an amino acid sequence thatappears in a humanized Lag-3 polypeptide of SEQ ID NO:8.

In some embodiments, an isolated non-human cell or tissue is provided,whose genome comprises a Lag-3 gene (or locus) as described herein. Insome embodiments, a cell is a lymphocyte. In some embodiments, a cell isselected from a B cell, dendritic cell, macrophage, monocyte, and a Tcell. In some embodiments, a tissue is selected from adipose, bladder,brain, breast, bone marrow, eye, heart, intestine, kidney, liver, lung,lymph node, muscle, pancreas, plasma, serum, skin, spleen, stomach,thymus, testis, ovum, and a combination thereof.

In some embodiments, an immortalized cell made, generated or producedfrom an isolated non-human cell as described herein is provided.

In some embodiments, a non-human embryonic stem (ES) cell is provided,whose genome comprises a Lag-3 gene (or locus) as described herein. Insome embodiments, a non-human embryonic stem cell is a rodent embryonicstem cell. In some certain embodiments, a rodent embryonic stem cell isa mouse embryonic stem cell and is from a 129 strain, C57BL strain, or amixture thereof. In some certain embodiments, a rodent embryonic stemcell is a mouse embryonic stem cell and is a mixture of 129 and C57BLstrains. In some embodiments, a non-human ES cell as described hereincomprises any one of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15 is provided.

In some embodiments, use of a non-human embryonic stem cell as describedherein to make a non-human animal is provided. In some certainembodiments, a non-human embryonic stem cell is a mouse embryonic stemcell and is used to make a mouse comprising a humanized Lag-3 gene (orlocus) as described herein. In some certain embodiments, a non-humanembryonic stem cell is a rat embryonic stem cell and is used to make arat comprising a humanized Lag-3 gene (or locus) as described herein.

In some embodiments, a non-human embryo comprising, made from, obtainedfrom, or generated from a non-human embryonic stem cell as describedherein is provided. In some certain embodiments, a non-human embryo is arodent embryo; in some embodiments, a mouse embryo; in some embodiments,a rat embryo.

In some embodiments, use of a non-human embryo described herein to makea non-human animal is provided. In some certain embodiments, a non-humanembryo is a mouse embryo and is used to make a mouse comprising ahumanized Lag-3 gene (or locus) as described herein. In some certainembodiments, a non-human embryo is a rat embryo and is used to make arat comprising a humanized Lag-3 gene (or locus) as described herein.

In some embodiments, a kit is provided, comprising an isolated non-humancell or tissue as described herein, an immortalized cell as describedherein, non-human embryonic stem cell as described herein, a non-humanembryo as described herein, or a non-human animal as described herein.

In some embodiments, a kit as described herein, for use in themanufacture and/or development of a drug (e.g., an antibody orantigen-binding fragment thereof) for therapy or diagnosis is provided.

In some embodiments, a kit as described herein, for use in themanufacture and/or development of a drug (e.g., an antibody orantigen-binding fragment thereof) for the treatment, prevention oramelioration of a disease, disorder or condition is provided.

In some embodiments, a transgene, nucleic acid construct, DNA construct,or targeting vector as described herein is provided. In some certainembodiments, a transgene, nucleic acid construct, DNA construct, ortargeting vector comprises a Lag-3 gene (or locus), in whole or in part,as described herein. In some certain embodiments, a transgene, nucleicacid construct, DNA construct, or targeting vector comprises a DNAfragment that includes a Lag-3 gene (or locus), in whole or in part, asdescribed herein. In some certain embodiments, a transgene, nucleic acidconstruct, DNA construct, or targeting vector comprises a Lag-3 gene (orlocus) that comprises any one of SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15. Insome certain embodiments, a transgene, nucleic acid construct, DNAconstruct, or targeting vector further comprises one or more selectionmarkers. In some certain embodiments, a transgene, nucleic acidconstruct, DNA construct, or targeting vector further comprises one ormore site-specific recombination sites (e.g., loxP, Frt, or combinationsthereof). In some certain embodiments, a transgene, nucleic acidconstruct, DNA construct, or targeting vector is depicted in FIG. 3.

In some embodiments, use of a transgene, nucleic acid construct, DNAconstruct, or targeting vector as described herein to make a non-humanembryonic stem cell, non-human cell, non-human embryo and/or non-humananimal is provided.

In some embodiments, a method of making a non-human animal thatexpresses a Lag-3 polypeptide from an endogenous Lag-3 gene is provided,wherein the Lag-3 polypeptide comprises a human sequence, the methodcomprising (a) placing a genomic fragment into an endogenous Lag-3 genein a non-human embryonic stem cell, said genomic fragment comprising anucleotide sequence that encodes a human Lag-3 polypeptide in whole orin part; (b) obtaining the non-human embryonic stem cell generated in(a); and, (c) creating a non-human animal using the non-human embryonicstem cell of (b).

In some embodiments of a method of making a non-human animal thatexpresses a Lag-3 polypeptide from an endogenous Lag-3 gene, the methodfurther comprises a step of placing a genomic fragment into anendogenous Pdcd1 gene of the non-human embryonic stem cell of (a), saidgenomic fragment comprising a nucleotide sequence that encodes a humanPD-1 polypeptide in whole or in part. In some embodiments of a method ofmaking a non-human animal that expresses a Lag-3 polypeptide from anendogenous Lag-3 gene, a genomic fragment comprising a nucleotidesequence that encodes a human PD-1 polypeptide in whole or in part isplaced into an endogenous Pdcd1 gene of the non-human embryonic stemcell of (a) prior to, simultaneously with, or after the placement ofgenomic fragment that comprises the nucleotide sequence that encodes thehuman Lag-3 polypeptide in whole or in part into the endogenous Lag-3gene. In some embodiments of a method of making a non-human animal thatexpresses a Lag-3 polypeptide from an endogenous Lag-3 gene, the methodfurther comprises breeding the non-human animal of (c) with a secondnon-human animal, said second non-human animal having a genomecomprising a Pdcd1 gene that encodes a PD-1 polypeptide comprising ahuman portion and an endogenous portion.

In some embodiments, a nucleotide sequence comprises human Lag-3 exons2-4. In some embodiments, a nucleotide sequence encodes at least aminoacids 29-260 (or 23-260 or 21-260) of a human LAG-3 polypeptide. In someembodiments, a nucleotide sequence comprises one or more selectionmarkers. In some embodiments, a nucleotide sequence comprises one ormore site-specific recombination sites.

In some embodiments, a method of making a non-human animal whose genomecomprises a Lag-3 gene that encodes a Lag-3 polypeptide having a humanportion and an endogenous portion is provided, the method comprisingmodifying the genome of a non-human animal so that it comprises a Lag-3gene that encodes a Lag-3 polypeptide having a human portion and anendogenous portion, which portions are operably linked to a non-humanLag-3 promoter, thereby making said non-human animal.

In some embodiments of a method of making a non-human animal whosegenome comprises a Lag-3 gene that encodes a Lag-3 polypeptide having ahuman portion and an endogenous portion, a Lag-3 gene is modified toinclude exons 2-4 (or exons 2, 3 and 4) of a human LAG-3 gene. In someembodiments of a method of making a non-human animal whose genomecomprises a Lag-3 gene that encodes a Lag-3 polypeptide having a humanportion and an endogenous portion, a Lag-3 gene is modified to encode atleast amino acids 29-260 (or 23-260 or 21-260) of a human LAG-3polypeptide.

In some embodiments of a method of making a non-human animal whosegenome comprises a Lag-3 gene that encodes a Lag-3 polypeptide having ahuman portion and an endogenous portion, the method further comprisesmodifying the genome of the non-human animal so that it comprises aPdcd1 gene that encodes a PD-1 polypeptide comprising a human portionand an endogenous portion. In some embodiments of a method of making anon-human animal whose genome comprises a Lag-3 gene that encodes aLag-3 polypeptide having a human portion and an endogenous portion,modifying the genome of the non-human animal so that it comprises aPdcd1 gene that encodes a PD-1 polypeptide comprising a human portionand an endogenous portion is performed prior to, simultaneously with, orafter modifying the genome of the non-human animal so that it comprisesa Lag-3 gene that encodes a Lag-3 polypeptide having a human portion andan endogenous portion. In some embodiments of a method of making anon-human animal whose genome comprises a Lag-3 gene that encodes aLag-3 polypeptide having a human portion and an endogenous portion, themethod further comprises breeding the non-human animal whose genomecomprises a Lag-3 gene that encodes a Lag-3 polypeptide having a humanportion and an endogenous portion with a second non-human animal, saidsecond non-human animal having a genome comprising a Pdcd1 gene thatencodes a PD-1 polypeptide comprising a human portion and an endogenousportion.

In some embodiments, a non-human animal obtainable by (made from,obtained from, or generated from) any one of the methods as describedherein is provided.

In some embodiments, a method of assessing anti-tumor efficacy of a drugtargeting human LAG-3 is provided, the method comprising the steps ofadministering the drug to a non-human animal as described herein, andperforming an assay to determine one or more anti-tumor properties ofthe drug targeting human LAG-3.

In some embodiments, a method of assessing the pharmacokineticproperties of a drug targeting human LAG-3 is provided, the methodcomprising the steps of administering the drug to a non-human animal asdescribed herein, and performing an assay to determine one or morepharmacokinetic properties of the drug targeting human LAG-3.

In some embodiments, a drug targeting human LAG-3 is a Lag-3 antagonist.In some embodiments, a drug targeting human LAG-3 is a Lag-3 agonist. Insome embodiments, a drug targeting human LAG-3 is an anti-Lag-3antibody.

In some embodiments, a drug targeting human LAG-3 is administered to anon-human animal as described herein intravenously, intraperitoneally orsubcutaneously.

In some embodiments, a non-human animal is provided, whose genomecomprises a Lag-3 gene that includes an endogenous portion thatcomprises endogenous Lag-3 exons 1, 5, 6, 7 and 8; and a human portionthat comprises exons 2-4 (or exons 2, 3 and 4) of a human LAG-3 gene;wherein the endogenous and human portions are operably linked to anendogenous non-human Lag-3 promoter, and wherein the non-human animalexpresses a Lag-3 polypeptide that comprises amino acids 29-260 (or23-260 or 21-260) of a human LAG-3 polypeptide.

In some embodiments, a non-human animal tumor model is provided, whichnon-human animal expresses a Lag-3 and/or PD-1 polypeptide as describedherein.

In some embodiments, a non-human animal tumor model is provided, whichnon-human animal has a genome comprising a Lag-3 and/or Pdcd1 gene asdescribed herein.

In some embodiments, a non-human animal tumor model is provided,obtained by (a) providing a non-human animal whose genome comprises aLag-3 and/or Pdcd1 gene as described herein; and (b) implanting one ormore tumor cells in the non-human animal of (a); thereby providing saidnon-human animal tumor model.

In some embodiments, a non-human animal or cell as described herein isprovided, for use in the manufacture and/or development of a drug fortherapy or diagnosis.

In some embodiments, a non-human animal or cell as described herein isprovided, for use in the manufacture of a medicament for the treatment,prevention or amelioration of a disease, disorder or condition.

In some embodiments, use of a non-human animal or cell as describedherein in the manufacture and/or development of a drug or vaccine foruse in medicine, such as use as a medicament is provided.

In some embodiments, use of a non-human animal or cell as describedherein in the manufacture of a medicament for the treatment of adisease, disorder or condition is provided.

In some embodiments, use of a non-human animal or cell as describedherein in the manufacture and/or development of an antibody that bindsan immune checkpoint molecule is provided.

In some embodiments, a disease, disorder or condition as describedherein is cancer or a neoplasm. In some embodiments, a disease, disorderor condition as described herein is an autoimmune (or inflammatory)disease, disorder or condition. In some embodiments, a disease, disorderor condition as described herein is an infectious disease, disorder orcondition.

In various embodiments, a non-human Lag-3 promoter is or comprises anendogenous non-human Lag-3 promoter.

In various embodiments, a human portion (or human sequence, ornucleotide sequence) of Lag-3 or Pdcd1 gene as described herein is orcomprises a sequence that is codon-optimized for expression in anon-human animal (or non-human cell or tissue) as described herein.

In various embodiments, a non-human animal (or non-human cell or tissue,or non-human embryonic stem cell, or non-human embryo) as describedherein further expresses a humanized PD-1 polypeptide, which humanizedPD-1 polypeptide comprises a human portion and an endogenous portion. Insome embodiments, a human portion of a PD-1 polypeptide comprisessubstantially the extracellular domain of a human PD-1 polypeptide; andin specific embodiments, a human portion comprises amino acids 21-170,26-169, 27-169, 27-145 or 35-145 of a human PD-1 polypeptide. In someembodiments, an endogenous portion of a PD-1 polypeptide comprises anintracellular portion and/or a transmembrane portion of an endogenousPD-1 polypeptide; in some embodiments, an endogenous portion of a PD-1polypeptide comprises the intracellular domain of an endogenous PD-1polypeptide, and in certain embodiments, an endogenous portion of a PD-1polypeptide further comprises substantially the transmembrane domain ofan endogenous PD-1 polypeptide.

In various embodiments, the genome of a non-human animal (or non-humancell or tissue, or non-human embryonic stem cell, or non-human embryo)as described herein further comprises a Pdcd1 gene that comprises anendogenous portion and a human portion, wherein the endogenous and humanportions are operably linked to a non-human Pdcd1 promoter. In someembodiments, an endogenous portion of a Pdcd1 gene comprises endogenousPdcd1 exons 1, 4 and 5. In some embodiments, an endogenous portion of aPdcd1 gene further comprises endogenous Pdcd1 exon 3 in whole or inpart, for example, a 3′ portion of endogenous exon 3 that encodes aminoacids that are part of the transmembrane domain of an endogenous PD-1polypeptide. In some embodiments, a human portion of a Pdcd1 geneencodes amino acids 35-145, 27-145, 27-169, 26-169 or 21-170 of a humanPD-1 polypeptide. In some embodiments, a human portion of a Pdcd1 genecomprises exon 2 of a human PDCD1 gene; in some certain embodiments,further comprises a human PDCD1 exon 3 in whole or in part, for example,a 5′ portion of human exon 3 that encodes amino acids that are part ofthe extracellular domain of a human PD-1 polypeptide. In specificembodiments, a humanized Pdcd1 gene in a non-human animal comprises exon1 of an endogenous Pdcd1 gene of the non-human animal, exon 2 and a partof exon 3 (e.g., a 5′ portion of exon 3 that encodes amino acids thatare part of the extracellular domain) of a human PDCD1 gene, followed bya part of exon 3 (e.g., a 3′ portion of exon 3 that encodes amino acidsthat are part of the transmembrane domain) and exons 4-5 of anendogenous non-human Pdcd1 gene.

In various embodiments, a non-human Pdcd1 promoter is or comprises anendogenous non-human Pdcd1 promoter.

In various embodiments, a human portion of a Lag-3 gene encodes an aminoacid sequence of immunoglobulin-like (Ig-like) domains 1 (D1) and 2 (D2)of a human LAG-3 polypeptide, or encodes an amino acid sequence of ahuman LAG-3 polypeptide that is responsible for binding MHC II.

In various embodiments, a human portion of a Lag-3 polypeptide comprisesan amino acid sequence of immunoglobulin-like (Ig-like) domains 1 (D1)and 2 (D2) of a human LAG-3 polypeptide, or comprises an amino acidsequence of a human LAG-3 polypeptide that is responsible for bindingMHC II.

In various embodiments, a non-human animal as described herein is arodent; in some embodiments, a mouse; in some embodiments, a rat. Insome embodiments, a mouse as described herein is selected from the groupconsisting of a 129 strain, a BALB/C strain, a C57BL/6 strain, and amixed 129xC57BL/6 strain; in some certain embodiments, a C57BL/6 strain.

As used in this application, the terms “about” and “approximately” areused as equivalents. Any numerals used in this application with orwithout about/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art.

BRIEF DESCRIPTION OF THE DRAWING

The Drawing included herein, which is composed of the following Figures,is for illustration purposes only and not for limitation.

FIG. 1 shows a diagram, not to scale, of the genomic organization of anon-human (e.g., mouse) and human Lymphocyte-activation gene 3 (Lag-3).Exons are numbered above or below each exon. Untranslated regions (openboxes) are also indicated for each gene Immunoglobulin-like domains areindicated, not to scale, by stripped boxes and the symbolic abbreviation“Ig” above the encoding exons.

FIG. 2 shows an alignment of representative amino acid sequences ofhuman LAG-3 (hLAG3) (SEQ ID NO: 6), mouse Lag-3 (mLag3) (SEQ ID NO: 4)and humanized Lag-3 (HumLAG3) (SEQ ID NO: 8). Asterisks indicateimmunoglobulin-like (Ig-like) domains and underlined text indicatesamino acids encoded by inserted human LAG-3 exons (i.e., exons 2, 3 and4). Ig-like domains 1 (D1) and 2 (D2) are separated by a single aminoindicated by forward slash below the sequence.

FIG. 3 shows a diagram, not to scale, of an exemplary method forhumanization of a non-human Lymphocyte-activation gene 3 (Lag-3).Selected nucleotide junction locations are marked with a line below eachjunction and each indicated by SEQ ID NO.

FIG. 4 shows a diagram, not to scale, of the genomic organization of amouse and human Lymphocyte-activation gene 3 (Lag-3) indicating theapproximate locations of probes employed in an assay described inExample 1.

FIG. 5 shows representative histograms of activated splenocytes fromwild type C57BL/6 (wild type), homozygous humanized Lag-3 (HumLAG-3),homozygous humanized PD-1 (HumPD-1), and homozygous double humanizedLag-3xPD-1 (HumPD-1xLAG-3) mice stained with antibodies to human LAG-3,human PD-1, mouse Lag-3, and mouse PD-1 or stained with respectiveisotype control antibody. Positive staining is indicated by filledcurves, while staining with isotype control antibodies is indicated byunfilled curves. All staining profiles represent cells within CD4⁺gating. Medium florescent intensity (MFI) with antibody staining on CD4⁺T cells is indicated for each histogram (top row: human LAG-3expression; 2nd row: mouse Lag-3 expression; 3rd row: human PD-1expression; bottom row: mouse PD-1 expression). Genotype of mice isindicated at the top of each column. For humanized PD-1 mice see U.S.patent application Ser. No. 14/744,592, filed 19 Jun. 2015, andInternational Patent Application No. PCT/US15/036649, filed 19 Jun.2015; both of which are incorporated herein by reference.

FIG. 6 shows representative average tumor volume (mm³±SEM) over 35 daysin various treatment groups of double humanized Lag-3/PD-1 mice(control, circle: human isotype control antibody not specific for mouseor human LAG-3 or mouse or human PD-1; anti-LAG3, diamond: anti-humanLAG-3 antibody; anti-PD1, hexagon: anti-human PD-1 antibody;). Arrowsindicate the days of antibody treatment.

FIG. 7 shows average tumor volumes (mm³±SEM) in various treatment groupsof double humanized Lag-3/PD-1 mice at multiple time points post tumorimplantation in an experiment to assess efficacy of anti-LAG3 antibodyalone and in combination with anti-human PD-1 antibody againstestablished MC38 tumors (control, circle: human isotype control antibodynot specific for mouse or human LAG-3 or mouse or human PD-1; anti-LAG3,diamond: anti-human LAG-3 antibody; anti-PD1, hexagon: anti-human PD-1antibody; anti-LAG3+anti-PD-1, triangle: combination of anti-human LAG-3antibody and anti-human PD-1 antibody). Treatment days are indicated byarrows.

FIG. 8 shows exemplary rodent (e.g., rat and mouse), human, andhumanized lymphocyte-activation gene 3 (Lag-3) sequences, as well as anexemplary synthetic DNA fragment for humanization of a non-human Lag-3gene. For mRNA sequences, bold font indicates coding sequence, andconsecutive exons, where indicated, are separated by alternatingunderlined text; for humanized mRNA sequences, human sequences arecontained within parentheses. For amino acid sequences, transmembranesequences are indicated by underlined font; for humanized amino acidsequences, human sequences are indicated in bold font and containedwithin parentheses.

FIGS. 9A-9D show junction sequences in certain exemplified humanizedLag-3 locus. FIG. 9A shows the nucleotide sequence (SEQ ID NO: 12)across the upstream insertion point, which indicates endogenous mousesequence (contained within the parentheses below) contiguous with humanLAG-3 genomic sequence at the insertion point. FIG. 9B shows thenucleotide sequence (SEQ ID NO: 13) across the 5′ end of theself-deleting neomycin cassette, which indicates human LAG-3 genomicsequence contiguous with cassette sequence (contained within theparentheses below with a SalI-XhoI compatible end italicized and a loxPsequence in bold font) downstream of the insertion point. FIG. 9C showsthe nucleotide sequence (SEQ ID NO: 14) across the downstream insertionpoint at the 3′ end of the self-deleting neomycin cassette, whichindicates cassette sequence (contained within the parentheses below witha loxP site in bold, an I-CeuI recognition site underlined and an NheIrecognition site italicized) contiguous with mouse Lag-3 genomicsequence.

FIG. 9D shows the nucleotide sequence (SEQ ID NO: 15) across theupstream insertion point after deletion of the neomycin cassette (77 bpremaining in intron 4), which indicates mouse and human genomic sequencejuxtaposed with remaining cassette sequence loxP sequence (containedwithin the parentheses below with a SalI-XhoI compatible end italicized,a loxP site in bold, an I-CeuI restriction site underlined and an NheIrestriction site italicized).

DEFINITIONS

This invention is not limited to particular methods and experimentalconditions described herein, as such methods and conditions may vary. Itis also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention is defined bythe claims.

Unless defined otherwise, all terms and phrases used herein include themeanings that the terms and phrases have attained in the art, unless thecontrary is clearly indicated or clearly apparent from the context inwhich the term or phrase is used. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, particular methods andmaterials are now described. All patent and non-patent publicationsmentioned herein are hereby incorporated by reference.

Approximately: as applied herein to one or more values of interest,includes a value that is similar to a stated reference value. In certainembodiments, the term “approximately” or “about” includes a range ofvalues that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in eitherdirection (greater than or less than) of the stated reference valueunless otherwise stated or otherwise evident from the context (exceptwhere such number would exceed 100% of a possible value).

Biologically active: as used herein, includes a characteristic of anyagent that has activity in a biological system, in vitro or in vivo(e.g., in an organism). For instance, an agent that, when present in anorganism, has a biological effect within that organism, is considered tobe biologically active. In particular embodiments, where a protein orpolypeptide is biologically active, a portion of that protein orpolypeptide that shares at least one biological activity of the proteinor polypeptide is typically referred to as a “biologically active”portion.

Comparable: as used herein, includes two or more agents, entities,situations, sets of conditions, etc. that may not be identical to oneanother but that are sufficiently similar to permit comparison betweenthem so that conclusions may reasonably be drawn based on differences orsimilarities observed. Those of ordinary skill in the art willunderstand, in context, what degree of identity is required in any givencircumstance for two or more such agents, entities, situations, sets ofconditions, etc. to be considered comparable.

Conservative: as used herein to describe a conservative amino acidsubstitution, includes substitution of an amino acid residue by anotheramino acid residue having a side chain R group with similar chemicalproperties (e.g., charge or hydrophobicity). In general, a conservativeamino acid substitution will not substantially change the functionalproperties of interest of a protein, for example, the ability of areceptor to bind to a ligand. Examples of groups of amino acids thathave side chains with similar chemical properties include: aliphaticside chains such as glycine, alanine, valine, leucine, and isoleucine;aliphatic-hydroxyl side chains such as serine and threonine;amide-containing side chains such as asparagine and glutamine; aromaticside chains such as phenylalanine, tyrosine, and tryptophan; basic sidechains such as lysine, arginine, and histidine; acidic side chains suchas aspartic acid and glutamic acid; and, sulfur-containing side chainssuch as cysteine and methionine. Conservative amino acids substitutiongroups include, for example, valine/leucine/isoleucine,phenylalanine/tyrosine, lysine/arginine, alanine/valine,glutamate/aspartate, and asparagine/glutamine. In some embodiments, aconservative amino acid substitution can be a substitution of any nativeresidue in a protein with alanine, as used in, for example, alaninescanning mutagenesis. In some embodiments, a conservative substitutionis made that has a positive value in the PAM250 log-likelihood matrixdisclosed in Gonnet et al. (1992) Exhaustive Matching of the EntireProtein Sequence Database, Science 256:1443-45, hereby incorporated byreference. In some embodiments, the substitution is a moderatelyconservative substitution wherein the substitution has a nonnegativevalue in the PAM250 log-likelihood matrix.

Control: as used herein, includes the art-understood meaning of a“control” being a standard against which results are compared.Typically, controls are used to augment integrity in experiments byisolating variables in order to make a conclusion about such variables.In some embodiments, a control is a reaction or assay that is performedsimultaneously with a test reaction or assay to provide a comparator. Asused herein, a “control” may include a “control animal”. A “controlanimal” may have a modification as described herein, a modification thatis different as described herein, or no modification (i.e., a wild-typeanimal). In one experiment, the “test” (i.e., the variable being tested)is applied. In the second experiment, the “control” (i.e., the variablebeing tested) is not applied. In some embodiments, a control is ahistorical control (i.e., of a test or assay performed previously, or anamount or result that is previously known). In some embodiments, acontrol is or comprises a printed or otherwise saved record. A controlmay be a positive control or a negative control.

Disruption: as used herein, includes the result of a homologousrecombination event with a DNA molecule (e.g., with an endogenoushomologous sequence such as a gene or gene locus). In some embodiments,a disruption may achieve or represent an insertion, deletion,substitution, replacement, missense mutation, or a frame-shift of a DNAsequence(s), or any combination thereof. Insertions may include theinsertion of entire genes or fragments of genes, e.g., exons, which maybe of an origin other than the endogenous sequence (e.g., a heterologoussequence). In some embodiments, a disruption may increase expressionand/or activity of a gene or gene product (e.g., of a protein encoded bya gene). In some embodiments, a disruption may decrease expressionand/or activity of a gene or gene product. In some embodiments, adisruption may alter sequence of a gene or an encoded gene product(e.g., an encoded protein). In some embodiments, a disruption maytruncate or fragment a gene or an encoded gene product (e.g., an encodedprotein). In some embodiments, a disruption may extend a gene or anencoded gene product; in some such embodiments, a disruption may achieveassembly of a fusion protein. In some embodiments, a disruption mayaffect level but not activity of a gene or gene product. In someembodiments, a disruption may affect activity but not level of a gene orgene product. In some embodiments, a disruption may have no significanteffect on level of a gene or gene product. In some embodiments, adisruption may have no significant effect on activity of a gene or geneproduct. In some embodiments, a disruption may have no significanteffect on either level or activity of a gene or gene product.

Determining, measuring, evaluating, assessing, assaying and analyzing:are used interchangeably herein to include any form of measurement, andinclude determining if an element is present or not. These terms includeboth quantitative and/or qualitative determinations. Assaying may berelative or absolute. “Assaying for the presence of” can be determiningthe amount of something present and/or determining whether or not it ispresent or absent.

Endogenous locus or endogenous gene: as used herein, includes a geneticlocus found in a parent or reference organism prior to introduction ofan alteration, disruption, deletion, insertion, modification,replacement, or substitution as described herein. In some embodiments,the endogenous locus has a sequence found in nature. In someembodiments, the endogenous locus is a wild type locus. In someembodiments, the reference organism is a wild type organism. In someembodiments, the reference organism is an engineered organism. In someembodiments, the reference organism is a laboratory-bred organism(whether wild type or engineered).

Endogenous promoter: as used herein, includes a promoter that isnaturally associated, e.g., in a wild-type organism, with an endogenousgene.

Engineered: as used herein, in general, includes the aspect of havingbeen manipulated by the hand of man. For example, in some embodiments, apolynucleotide may be considered to be “engineered” when two or moresequences that are not linked together in that order in nature aremanipulated by the hand of man to be directly linked to one another inthe engineered polynucleotide. In some particular such embodiments, anengineered polynucleotide may comprise a regulatory sequence that isfound in nature in operative association with a first coding sequencebut not in operative association with a second coding sequence, islinked by the hand of man so that it is operatively associated with thesecond coding sequence. Alternatively or additionally, in someembodiments, first and second nucleic acid sequences that each encodepolypeptide elements or domains that in nature are not linked to oneanother may be linked to one another in a single engineeredpolynucleotide. Comparably, in some embodiments, a cell or organism maybe considered to be “engineered” if it has been manipulated so that itsgenetic information is altered (e.g., new genetic material notpreviously present has been introduced, or previously present geneticmaterial has been altered or removed). As is common practice and isunderstood by those in the art, progeny of an engineered polynucleotideor cell are typically still referred to as “engineered” even though theactual manipulation was performed on a prior entity. Furthermore, aswill be appreciated by those skilled in the art, a variety ofmethodologies are available through which “engineering” as describedherein may be achieved. For example, in some embodiments, “engineering”may involve selection or design (e.g., of nucleic acid sequences,polypeptide sequences, cells, tissues, and/or organisms) through use ofcomputer systems programmed to perform analysis or comparison, orotherwise to analyze, recommend, and/or select sequences, alterations,etc.). Alternatively or additionally, in some embodiments, “engineering”may involve use of in vitro chemical synthesis methodologies and/orrecombinant nucleic acid technologies such as, for example, nucleic acidamplification (e.g., via the polymerase chain reaction) hybridization,mutation, transformation, transfection, etc., and/or any of a variety ofcontrolled mating methodologies. As will be appreciated by those skilledin the art, a variety of established such techniques (e.g., forrecombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection, etc.)) are wellknown in the art and described in various general and more specificreferences that are cited and/or discussed throughout the presentspecification. See e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989).

Gene: as used herein, includes a DNA sequence in a chromosome that codesfor a product (e.g., an RNA product and/or a polypeptide product). Insome embodiments, a gene includes coding sequence (i.e., sequence thatencodes a particular product). In some embodiments, a gene includesnon-coding sequence. In some particular embodiments, a gene may includeboth coding (e.g., exonic) and non-coding (e.g., intronic) sequence. Insome embodiments, a gene may include one or more regulatory sequences(e.g., promoters, enhancers, etc.) and/or intron sequences that, forexample, may control or impact one or more aspects of gene expression(e.g., cell-type-specific expression, inducible expression, etc.). Forthe purpose of clarity we note that, as used in the present application,the term “gene” generally includes a portion of a nucleic acid thatencodes a polypeptide; the term may optionally encompass regulatorysequences, as will be clear from context to those of ordinary skill inthe art. This definition is not intended to exclude application of theterm “gene” to non-protein-coding expression units but rather to clarifythat, in most cases, the term as used in this document includes apolypeptide-coding nucleic acid.

Heterologous: as used herein, includes an agent or entity from adifferent source. For example, when used in reference to a polypeptide,gene, or gene product present in a particular cell or organism, the termclarifies that the relevant polypeptide, gene, or gene product: 1) wasengineered by the hand of man; 2) was introduced into the cell ororganism (or a precursor thereof) through the hand of man (e.g., viagenetic engineering); and/or 3) is not naturally produced by or presentin the relevant cell or organism (e.g., the relevant cell type ororganism type).

Host cell: as used herein, includes a cell into which a heterologous(e.g., exogenous) nucleic acid or protein has been introduced. Personsof skill upon reading this disclosure will understand that such termsinclude not only the particular subject cell, but also is used toinclude the progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. In some embodiments, a host cell is or comprises aprokaryotic or eukaryotic cell. In general, a host cell is any cell thatis suitable for receiving and/or producing a heterologous nucleic acidor protein, regardless of the Kingdom of life to which the cell isdesignated. Exemplary cells include those of prokaryotes and eukaryotes(single-cell or multiple-cell), bacterial cells (e.g., strains of E.coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria cells,fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris,P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21,baculovirus-infected insect cells, Trichoplusia ni, etc.), non-humananimal cells, human cells, or cell fusions such as, for example,hybridomas or quadromas. In some embodiments, the cell is a human,monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cellis eukaryotic and is selected from the following cells: CHO (e.g., CHOK1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1,kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2,WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431(epidermal), CV-1, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT060562, Sertoli cell, BRL 3A cell, HT1080 cell, myeloma cell, tumorcell, and a cell line derived from an aforementioned cell. In someembodiments, the cell comprises one or more viral genes, e.g., a retinalcell that expresses a viral gene (e.g., a PER.C6™ cell). In someembodiments, a host cell is or comprises an isolated cell. In someembodiments, a host cell is part of a tissue. In some embodiments, ahost cell is part of an organism.

Humanized: is used herein in accordance with its art-understood meaningto include nucleic acids or proteins whose structures (i.e., nucleotideor amino acid sequences) include portions that correspond substantiallyor identically with structures of a particular gene or protein found innature in a non-human animal, and also include portions that differ fromthat found in the relevant particular non-human gene or protein andinstead correspond more closely with comparable structures found in acorresponding human gene or protein. In some embodiments, a “humanized”gene is one that encodes a polypeptide having substantially the aminoacid sequence as that of a human polypeptide (e.g., a human protein orportion thereof—e.g., characteristic portion thereof). To give but oneexample, in the case of a membrane receptor, a “humanized” gene mayencode a polypeptide having an extracellular portion, in whole or inpart, having an amino acid sequence as that of a human extracellularportion and the remaining sequence as that of a non-human (e.g., mouse)polypeptide. In some embodiments, a humanized gene comprises at least aportion of a DNA sequence of a human gene. In some embodiments, ahumanized gene comprises an entire DNA sequence of a human gene. In someembodiments, a humanized protein comprises a sequence having a portionthat appears in a human protein. In some embodiments, a humanizedprotein comprises an entire sequence of a human protein and is expressedfrom an endogenous locus of a non-human animal that corresponds to thehomolog or ortholog of the human gene.

Identity: as used herein in connection with a comparison of sequences,includes identity as determined by a number of different algorithmsknown in the art that can be used to measure nucleotide and/or aminoacid sequence identity. In some embodiments, identities as describedherein are determined using a ClustalW v. 1.83 (slow) alignmentemploying an open gap penalty of 10.0, an extend gap penalty of 0.1, andusing a Gonnet similarity matrix (MACVECTOR™ 10.0.2, MacVector Inc.,2008).

In vitro: as used herein includes events that occur in an artificialenvironment, e.g., in a test tube or reaction vessel, in cell culture,etc., rather than within a multi-cellular organism.

In vivo: as used herein includes events that occur within amulti-cellular organism, such as a human and a non-human animal. In thecontext of cell-based systems, the term may be used to include eventsthat occur within a living cell (as opposed to, for example, in vitrosystems).

Isolated: as used herein, includes a substance and/or entity that hasbeen (1) separated from at least some of the components with which itwas associated when initially produced (whether in nature and/or in anexperimental setting), and/or (2) designed, produced, prepared, and/ormanufactured by the hand of man. Isolated substances and/or entities maybe separated from about 10%, about 20%, about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than about 99% of the other components with which they wereinitially associated. In some embodiments, isolated agents are about80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, about 99%, or more thanabout 99% pure. As used herein, a substance is “pure” if it issubstantially free of other components. In some embodiments, as will beunderstood by those skilled in the art, a substance may still beconsidered “isolated” or even “pure”, after having been combined withcertain other components such as, for example, one or more carriers orexcipients (e.g., buffer, solvent, water, etc.); in such embodiments,percent isolation or purity of the substance is calculated withoutincluding such carriers or excipients. To give but one example, in someembodiments, a biological polymer such as a polypeptide orpolynucleotide that occurs in nature is considered to be “isolated”when: a) by virtue of its origin or source of derivation is notassociated with some or all of the components that accompany it in itsnative state in nature; b) it is substantially free of otherpolypeptides or nucleic acids of the same species from the species thatproduces it in nature; or c) is expressed by or is otherwise inassociation with components from a cell or other expression system thatis not of the species that produces it in nature. Thus, for instance, insome embodiments, a polypeptide that is chemically synthesized or issynthesized in a different cellular system from that which produces itin nature is considered to be an “isolated” polypeptide. Alternativelyor additionally, in some embodiments, a polypeptide that has beensubjected to one or more purification techniques may be considered to bean “isolated” polypeptide to the extent that it has been separated fromother components: a) with which it is associated in nature; and/or b)with which it was associated when initially produced.

“Locus” or “Loci”: as used herein, includes a specific location(s) of agene (or significant sequence), DNA sequence, polypeptide-encodingsequence, or position on a chromosome of the genome of an organism. Forexample, a “Lag-3 locus” may include the specific location of a Lag-3gene, Lag-3 DNA sequence, Lag-3-encoding sequence, or Lag-3 position ona chromosome of the genome of an organism that has been identified as towhere such a sequence resides. A “Lag-3 locus” may comprise a regulatoryelement of a Lag-3 gene, including, but not limited to, an enhancer, apromoter, 5′ and/or 3′ UTR, or a combination thereof. Those of ordinaryskill in the art will appreciate that chromosomes may, in someembodiments, contain hundreds or even thousands of genes and demonstratephysical co-localization of similar genetic loci when comparing betweendifferent species. Such genetic loci may be described as having sharedsynteny.

Non-human animal: as used herein, includes any vertebrate organism thatis not a human. In some embodiments, a non-human animal is a cyclostome,a bony fish, a cartilaginous fish (e.g., a shark or a ray), anamphibian, a reptile, a mammal, and a bird. In some embodiments, anon-human animal as described herein is a mammal. In some embodiments, anon-human mammal is a primate, a goat, a sheep, a pig, a dog, a cow, ora rodent. In some embodiments, a non-human animal as described herein isa small mammal, e.g., of the superfamily Dipodoidea or Muroidea. In someembodiments, a genetically modified animal as described herein is arodent. In some embodiments, a rodent as described herein is selectedfrom a mouse, a rat, and a hamster. In some embodiments, a rodent asdescribed herein is selected from the superfamily Muroidea. In someembodiments, a genetically modified animal as described herein is from afamily selected from Calomyscidae (e.g., mouse-like hamsters),Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae(true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae(climbing mice, rock mice, white-tailed rats, Malagasy rats and mice),Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., molerates, bamboo rats, and zokors). In some certain embodiments, agenetically modified rodent as described herein is selected from a truemouse or rat (family Muridae), a gerbil, a spiny mouse, and a crestedrat. In some certain embodiments, a genetically modified mouse asdescribed herein is from a member of the family Muridae. In someembodiment, a non-human animal as described herein is a rodent. In somecertain embodiments, a rodent as described herein is selected from amouse and a rat. In some embodiments, a non-human animal as describedherein is a mouse.

In some embodiments, a non-human animal as described herein is a rodentthat is a mouse of a C57BL strain selected from C57BL/A, C57BL/An,C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ,C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In some certainembodiments, a mouse as described herein is a 129 strain selected fromthe group consisting of a strain that is 129P1, 129P2, 129P3, 129X1,129S1 (e.g., 129S1/SV, 129S1/SvIm), 129S2, 129S4, 129S5, 129S9/SvEvH,129/SvJae, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g.,Festing et al., 1999, Mammalian Genome 10:836; Auerbach, W. et al.,2000, Biotechniques 29(5):1024-1028, 1030, 1032). In some certainembodiments, a genetically modified mouse as described herein is a mixof an aforementioned 129 strain and an aforementioned C57BL/6 strain. Insome certain embodiments, a mouse as described herein is a mix ofaforementioned 129 strains, or a mix of aforementioned BL/6 strains. Insome certain embodiments, a 129 strain of the mix as described herein isa 129S6 (129/SvEvTac) strain. In some embodiments, a mouse as describedherein is a BALB strain, e.g., BALB/c strain. In some embodiments, amouse as described herein is a mix of a BALB strain and anotheraforementioned strain.

In some embodiments, a non-human animal as described herein is a rat. Insome certain embodiments, a rat as described herein is selected from aWistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain,F344, F6, and Dark Agouti. In some certain embodiments, a rat strain asdescribed herein is a mix of two or more strains selected from the groupconsisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and DarkAgouti.

Nucleic acid: as used herein, in its broadest sense, includes anycompound and/or substance that is or can be incorporated into anoligonucleotide chain. In some embodiments, a “nucleic acid” is acompound and/or substance that is or can be incorporated into anoligonucleotide chain via a phosphodiester linkage. As will be clearfrom context, in some embodiments, “nucleic acid” includes individualnucleic acid residues (e.g., nucleotides and/or nucleosides); in someembodiments, “nucleic acid” includes an oligonucleotide chain comprisingindividual nucleic acid residues. In some embodiments, a “nucleic acid”is or comprises RNA; in some embodiments, a “nucleic acid” is orcomprises DNA. In some embodiments, a “nucleic acid” is, comprises, orconsists of one or more natural nucleic acid residues. In someembodiments, a “nucleic acid” is, comprises, or consists of one or morenucleic acid analogs. In some embodiments, a nucleic acid analog differsfrom a “nucleic acid” in that it does not utilize a phosphodiesterbackbone. For example, in some embodiments, a “nucleic acid” is,comprises, or consists of one or more “peptide nucleic acids”, which areknown in the art and have peptide bonds instead of phosphodiester bondsin the backbone, are considered within the scope of the presentinvention. Alternatively or additionally, in some embodiments, a“nucleic acid” has one or more phosphorothioate and/or5′-N-phosphoramidite linkages rather than phosphodiester bonds. In someembodiments, a “nucleic acid” is, comprises, or consists of one or morenatural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine,uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, anddeoxycytidine). In some embodiments, a “nucleic acid” is, comprises, orconsists of one or more nucleoside analogs (e.g., 2-aminoadenosine,2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine,2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases,intercalated bases, and combinations thereof). In some embodiments, a“nucleic acid” comprises one or more modified sugars (e.g.,2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose) ascompared with those in natural nucleic acids. In some embodiments, a“nucleic acid” has a nucleotide sequence that encodes a functional geneproduct such as an RNA or protein. In some embodiments, a “nucleic acid”includes one or more introns. In some embodiments, a “nucleic acid” isprepared by one or more of isolation from a natural source, enzymaticsynthesis by polymerization based on a complementary template (in vivoor in vitro), reproduction in a recombinant cell or system, and chemicalsynthesis. In some embodiments, a “nucleic acid” is at least 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900,1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residueslong. In some embodiments, a “nucleic acid” is single stranded; in someembodiments, a “nucleic acid” is double stranded. In some embodiments, a“nucleic acid” has a nucleotide sequence comprising at least one elementthat encodes, or is the complement of a sequence that encodes, apolypeptide. In some embodiments, a “nucleic acid” has enzymaticactivity.

Operably linked: as used herein, includes a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. “Operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest. The term “expression control sequence”, as used herein,includes polynucleotide sequences, which are necessary to effect theexpression and processing of coding sequences to which they are ligated.“Expression control sequences” include: appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism. For example, in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence, while in eukaryotes, typically,such control sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include componentswhose presence is essential for expression and processing, and can alsoinclude additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences.

Patient or subject: as used herein, includes any organism to which aprovided composition is or may be administered, e.g., for experimental,diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typicalpatients include animals (e.g., mammals such as mice, rats, rabbits,non-human primates, and/or humans). In some embodiments, a patient is anon-human animal. In some embodiments, a patient (e.g., a non-humananimal patient) may have a modification as described herein, amodification that is different as described herein or no modification(i.e., a wild-type non-human animal patient). In some embodiments, anon-human animal is suffering from or susceptible to one or moredisorders or conditions. In some embodiments, a non-human animaldisplays one or more symptoms of a disorder or condition. In someembodiments, a non-human animal has been diagnosed with one or moredisorders or conditions.

Polypeptide: as used herein, includes any polymeric chain of aminoacids. In some embodiments, a polypeptide has an amino acid sequencethat occurs in nature. In some embodiments, a polypeptide has an aminoacid sequence that does not occur in nature. In some embodiments, apolypeptide has an amino acid sequence that contains portions that occurin nature separately from one another (i.e., from two or more differentorganisms, for example, human and non-human portions). In someembodiments, a polypeptide has an amino acid sequence that is engineeredin that it is designed and/or produced through action of the hand ofman.

Recombinant: as used herein, is intended to include polypeptides (e.g.,Lag-3 polypeptides as described herein) that are designed, engineered,prepared, expressed, created or isolated by recombinant means, such aspolypeptides expressed using a recombinant expression vector transfectedinto a host cell, polypeptides isolated from a recombinant,combinatorial human polypeptide library (Hoogenboom H. R., 1997, TIBTech. 15:62-70; Azzazy H., and Highsmith W. E., 2002, Clin. Biochem.35:425-445; Gavilondo J. V., and Larrick J. W., 2002, BioTechniques29:128-145; Hoogenboom H., and Chames P., 2000, Immunology Today21:371-378), antibodies isolated from an animal (e.g., a mouse) that istransgenic for human immunoglobulin genes (see e.g., Taylor, L. D. etal., 1992, Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and Green L.L., 2002, Current Opinion in Biotechnology 13:593-597; Little M. et al.,2000, Immunology Today 21:364-370; Murphy, A. J. et al., 2014, Proc.Natl. Acad. Sci. U.S.A. 111(14):5153-5158) or polypeptides prepared,expressed, created or isolated by any other means that involves splicingselected sequence elements to one another. In some embodiments, one ormore of such selected sequence elements is found in nature. In someembodiments, one or more of such selected sequence elements is designedin silico. In some embodiments, one or more such selected sequenceelements result from mutagenesis (e.g., in vivo or in vitro) of a knownsequence element, e.g., from a natural or synthetic source. For example,in some embodiments, a recombinant polypeptide is comprised of sequencesfound in the genome of a source organism of interest (e.g., human,mouse, etc.). In some embodiments, a recombinant polypeptide has anamino acid sequence that resulted from mutagenesis (e.g., in vitro or invivo, for example in a non-human animal), so that the amino acidsequences of the recombinant polypeptides are sequences that, whileoriginating from and related to polypeptides sequences, may notnaturally exist within the genome of a non-human animal in vivo.

Replacement: as used herein, includes a process through which a“replaced” nucleic acid sequence (e.g., a gene) found in a host locus(e.g., in a genome) is removed from that locus, and a different,“replacement” nucleic acid is located in its place. In some embodiments,the replaced nucleic acid sequence and the replacement nucleic acidsequences are comparable to one another in that, for example, they arehomologous to one another and/or contain corresponding elements (e.g.,protein-coding elements, regulatory elements, etc.). In someembodiments, a replaced nucleic acid sequence includes one or more of apromoter, an enhancer, a splice donor site, a splice acceptor site, anintron, an exon, an untranslated region (UTR); in some embodiments, areplacement nucleic acid sequence includes one or more coding sequences.In some embodiments, a replacement nucleic acid sequence is a homolog ofthe replaced nucleic acid sequence. In some embodiments, a replacementnucleic acid sequence is an ortholog of the replaced sequence. In someembodiments, a replacement nucleic acid sequence is or comprises a humannucleic acid sequence. In some embodiments, including where thereplacement nucleic acid sequence is or comprises a human nucleic acidsequence, the replaced nucleic acid sequence is or comprises a rodentsequence (e.g., a mouse or rat sequence). The nucleic acid sequence soplaced may include one or more regulatory sequences that are part ofsource nucleic acid sequence used to obtain the sequence so placed(e.g., promoters, enhancers, 5′- or 3′-untranslated regions, etc.). Forexample, in various embodiments, the replacement is a substitution of anendogenous sequence with a heterologous sequence that results in theproduction of a gene product from the nucleic acid sequence so placed(comprising the heterologous sequence), but not expression of theendogenous sequence; the replacement is of an endogenous genomicsequence with a nucleic acid sequence that encodes a polypeptide thathas a similar function as a polypeptide encoded by the endogenoussequence (e.g., the endogenous genomic sequence encodes a Lag-3polypeptide, and the DNA fragment encodes one or more human Lag-3polypeptides, in whole or in part). In various embodiments, anendogenous gene or fragment thereof is replaced with a correspondinghuman gene or fragment thereof. A corresponding human gene or fragmentthereof is a human gene or fragment that is an ortholog of, or issubstantially similar or the same in structure and/or function, as theendogenous gene or fragment thereof that is replaced.

Reference: as used herein, describes a standard or control agent,cohort, individual, population, sample, sequence or value against whichan agent, animal, cohort, individual, population, sample, sequence orvalue of interest is compared. In some embodiments, a reference agent,cohort, individual, population, sample, sequence or value is testedand/or determined substantially simultaneously with the testing ordetermination of the agent, cohort, individual, population, sample,sequence or value of interest. In some embodiments, a reference agent,cohort, individual, population, sample, sequence or value is ahistorical reference, optionally embodied in a tangible medium. In someembodiments, a reference may refer to a control. As used herein, a“reference” may refer to a “reference animal”. A “reference animal” mayhave a modification as described herein, a modification that isdifferent as described herein or no modification (i.e., a wild-typeanimal) Typically, as would be understood by those skilled in the art, areference agent, animal, cohort, individual, population, sample,sequence or value is determined or characterized under conditionscomparable to those utilized to determine or characterize the agent,animal (e.g., a mammal), cohort, individual, population, sample,sequence or value of interest.

Substantially: as used herein, includes the qualitative condition ofexhibiting total or near-total extent or degree of a characteristic orproperty of interest. One of ordinary skill in the biological arts willunderstand that biological and chemical phenomena rarely, if ever, go tocompletion and/or proceed to completeness or achieve or avoid anabsolute result. The term “substantially” is therefore used herein tocapture the potential lack of completeness inherent in many biologicaland chemical phenomena. The term is also used herein when referring to asequence, a nucleic acid or protein molecule, or a protein domain, incomparison to a reference sequence, molecule or domain. For example, inreferring to a humanized Lag-3 polypeptide comprising substantially thesignal peptide of a non-human Lag-3 polypeptide, the phrase“substantially the signal peptide of a non-human Lag-3 polypeptide”includes a peptide that is substantially identical the signal peptide ofa non-human Lag-3 polypeptide, which peptide, in some embodiments, is atleast 85%, 90%, 95%, 95%, 99% or 100% identical in sequence with thesignal peptide of a non-human Lag-3 polypeptide; and in someembodiments, differs from the signal peptide of a non-human Lag-3polypeptide by not more than 5, 4, 3, 2 or 1 amino acid(s), preferablyonly at the N— or C— terminus of the signal peptide, e.g., by lackingamino acid(s) or having additional amino acid(s) at the at the N— or C—terminus of the signal peptide. As another example, in referring to ahumanized PD-1 polypeptide comprising substantially the extracellulardomain of a human PD-1 protein, the phrase “substantially theextracellular domain of a human PD-1 protein” includes a polypeptidethat is substantially identical with the extracellular domain of a humanPD-1 protein, which polypeptide, in some embodiments, is at least 85%,90%, 95%, 95%, 99% or 100% identical in sequence with the extracellulardomain of a human PD-1 protein; and in some embodiments, differs fromthe extracellular domain of a human PD-1 protein by not more than 5, 4,3, 2 or 1 amino acid(s), preferably only at the N— or C— terminus, e.g.,by lacking amino acids or having additional amino acids at the at the N—or C— terminus.

Substantial homology: as used herein, includes a comparison betweenamino acid or nucleic acid sequences. As will be appreciated by those ofordinary skill in the art, two sequences are generally considered to be“substantially homologous” if they contain homologous residues incorresponding positions. Homologous residues may be identical residues.Alternatively, homologous residues may be non-identical residues willappropriately similar structural and/or functional characteristics. Forexample, as is well known by those of ordinary skill in the art, certainamino acids are typically classified as “hydrophobic” or “hydrophilic”amino acids, and/or as having “polar” or “non-polar” side chains.Substitution of one amino acid for another of the same type may often beconsidered a “homologous” substitution. Typical amino acidcategorizations are summarized below:

Alanine Ala A Nonpolar Neutral 1.8 Arginine Arg R Polar Positive −4.5Asparagine Asn N Polar Neutral −3.5 Aspartic acid Asp D Polar Negative−3.5 Cysteine Cys C Nonpolar Neutral 2.5 Glutamic acid Glu E PolarNegative −3.5 Glutamine Gln Q Polar Neutral −3.5 Glycine Gly G NonpolarNeutral −0.4 Histidine His H Polar Positive −3.2 Isoleucine Ile INonpolar Neutral 4.5 Leucine Leu L Nonpolar Neutral 3.8 Lysine Lys KPolar Positive −3.9 Methionine Met M Nonpolar Neutral 1.9 PhenylalaninePhe F Nonpolar Neutral 2.8 Proline Pro P Nonpolar Neutral −1.6 SerineSer S Polar Neutral −0.8 Threonine Thr T Polar Neutral −0.7 TryptophanTrp W Nonpolar Neutral −0.9 Tyrosine Tyr Y Polar Neutral −1.3 Valine ValV Nonpolar Neutral 4.2 Ambiguous Amino Acids 3-Letter 1-LetterAsparagine or aspartic acid Asx B Glutamine or glutamic acid Glx ZLeucine or Isoleucine Xle J Unspecified or unknown amino acid Xaa X

As is well known in this art, amino acid or nucleic acid sequences maybe compared using any of a variety of algorithms, including thoseavailable in commercial computer programs such as BLASTN for nucleotidesequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acidsequences. Exemplary such programs are described in Altschul et al.,1990, Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410;Altschul et al., 1996, Methods Enzymol. 266:160-80; Altschul et al.,1997, Nucleic Acids Res. 25:3389-3402; Baxevanis et al., 1998Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins,Wiley; and Misener et al. (eds.) (1999) Bioinformatics Methods andProtocols (Methods in Molecular Biology, Vol. 132), Humana Press. Inaddition to identifying homologous sequences, the programs mentionedabove typically provide an indication of the degree of homology. In someembodiments, two sequences are considered to be substantially homologousif at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues arehomologous over a relevant stretch of residues. In some embodiments, therelevant stretch is a complete sequence. In some embodiments, therelevant stretch is at least 9, 10, 11, 12, 13, 14, 15, 16, 17 or moreresidues. In some embodiments, the relevant stretch includes contiguousresidues along a complete sequence. In some embodiments, the relevantstretch includes discontinuous residues along a complete sequence. Insome embodiments, the relevant stretch is at least 10, 15, 20, 25, 30,35, 40, 45, 50, or more residues.

Substantial identity: as used herein, includes a comparison betweenamino acid or nucleic acid sequences. As will be appreciated by those ofordinary skill in the art, two sequences are generally considered to be“substantially identical” if they contain identical residues incorresponding positions. As is well known in this art, amino acid ornucleic acid sequences may be compared using any of a variety ofalgorithms, including those available in commercial computer programssuch as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, andPSI-BLAST for amino acid sequences. Exemplary such programs aredescribed in Altschul et al., 1990, Basic local alignment search tool,J. Mol. Biol., 215(3): 403-410; Altschul et al., 1996, Methods Enzymol.266:160-80; Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402;Baxevanis et al., 1998, Bioinformatics: A Practical Guide to theAnalysis of Genes and Proteins, Wiley; Misener et al., (eds.) (1999)Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol.132), Humana Press. In addition to identifying identical sequences, theprograms mentioned above typically provide an indication of the degreeof identity. In some embodiments, two sequences are considered to besubstantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of theircorresponding residues are identical over a relevant stretch ofresidues. In some embodiments, the relevant stretch is a completesequence. In some embodiments, the relevant stretch is at least 10, 15,20, 25, 30, 35, 40, 45, 50, or more residues.

Targeting vector or targeting construct: as used herein, includes apolynucleotide molecule that comprises a targeting region. A targetingregion comprises a sequence that is identical or substantially identicalto a sequence in a target cell, tissue or animal and provides forintegration of the targeting construct into a position within the genomeof the cell, tissue or animal via homologous recombination. Targetingregions that target using site-specific recombinase recognition sites(e.g., loxP and/or Frt sites) are also included. In some embodiments, atargeting construct further comprises a nucleic acid sequence or gene ofparticular interest, a selectable marker, control and or regulatorysequences, and other nucleic acid sequences that allow for recombinationmediated through exogenous addition of proteins that aid in orfacilitate recombination involving such sequences. In some embodiments,a targeting construct further comprises a gene of interest in whole orin part, wherein the gene of interest is a heterologous gene thatencodes a protein, in whole or in part, that has a similar function as aprotein encoded by an endogenous sequence. In some embodiments, atargeting construct further comprises a humanized gene of interest, inwhole or in part, wherein the humanized gene of interest encodes aprotein, in whole or in part, that has a similar function as a proteinencoded by the endogenous sequence. In some embodiments, a targetingconstruct further comprises an engineered gene of interest, in whole orin part, wherein the engineered gene of interest encodes a protein, inwhole or in part, that has a similar function as a protein encoded by anendogenous sequence.

Variant: as used herein, includes an entity that shows significantstructural identity with a reference entity, but differs structurallyfrom the reference entity in the presence or level of one or morechemical moieties as compared with the reference entity. In manyembodiments, a “variant” also differs functionally from its referenceentity. In general, whether a particular entity is properly consideredto be a “variant” of a reference entity is based on its degree ofstructural identity with the reference entity. As will be appreciated bythose skilled in the art, any biological or chemical reference entityhas certain characteristic structural elements. A “variant”, bydefinition, is a distinct chemical entity that shares one or more suchcharacteristic structural elements. To give but a few examples, a smallmolecule may have a characteristic core structural element (e.g., amacrocycle core) and/or one or more characteristic pendent moieties sothat a variant of the small molecule is one that shares the corestructural element and the characteristic pendent moieties but differsin other pendent moieties and/or in types of bonds present (single vs.double, E vs. Z, etc.) within the core, a polypeptide may have acharacteristic sequence element comprised of a plurality of amino acidshaving designated positions relative to one another in linear orthree-dimensional space and/or contributing to a particular biologicalfunction, a nucleic acid may have a characteristic sequence elementcomprised of a plurality of nucleotide residues having designatedpositions relative to on another in linear or three-dimensional space.For example, a “variant polypeptide” may differ from a referencepolypeptide as a result of one or more differences in amino acidsequence and/or one or more differences in chemical moieties (e.g.,carbohydrates, lipids, etc.) covalently attached to the polypeptidebackbone. In some embodiments, a “variant polypeptide” shows an overallsequence identity with a reference polypeptide that is at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.Alternatively or additionally, in some embodiments, a “variantpolypeptide” does not share at least one characteristic sequence elementwith a reference polypeptide. In some embodiments, the referencepolypeptide has one or more biological activities. In some embodiments,a “variant polypeptide” shares one or more of the biological activitiesof the reference polypeptide. In some embodiments, a “variantpolypeptide” lacks one or more of the biological activities of thereference polypeptide. In some embodiments, a “variant polypeptide”shows a reduced level of one or more biological activities as comparedwith the reference polypeptide. In many embodiments, a polypeptide ofinterest is considered to be a “variant” of a parent or referencepolypeptide if the polypeptide of interest has an amino acid sequencethat is identical to that of the parent but for a small number ofsequence alterations at particular positions. Typically, fewer than 20%,15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the residues in the variantare substituted as compared with the parent. In some embodiments, a“variant” has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue ascompared with a parent. Often, a “variant” has a very small number(e.g., fewer than 5, 4, 3, 2, or 1) of substituted functional residues(i.e., residues that participate in a particular biological activity).Furthermore, a “variant” typically has not more than 5, 4, 3, 2, or 1additions or deletions, and often has no additions or deletions, ascompared with the parent. Moreover, any additions or deletions aretypically fewer than about 25, about 20, about 19, about 18, about 17,about 16, about 15, about 14, about 13, about 10, about 9, about 8,about 7, about 6, and commonly are fewer than about 5, about 4, about 3,or about 2 residues. In some embodiments, the parent or referencepolypeptide is one found in nature. As will be understood by those ofordinary skill in the art, a plurality of variants of a particularpolypeptide of interest may commonly be found in nature, particularlywhen the polypeptide of interest is an infectious agent polypeptide.

Vector: as used herein, includes a nucleic acid molecule capable oftransporting another nucleic acid to which it is associated. In someembodiment, vectors are capable of extra-chromosomal replication and/orexpression of nucleic acids to which they are linked in a host cell suchas a eukaryotic and/or prokaryotic cell. Vectors capable of directingthe expression of operatively linked genes are referred to herein as“expression vectors”.

Wild type: as used herein, has its art-understood meaning that includesan entity having a structure and/or activity as found in nature in a“normal” (as contrasted with mutant, diseased, altered, etc.) state orcontext. Those of ordinary skill in the art will appreciate that wildtype genes and polypeptides often exist in multiple different forms(e.g., alleles).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present invention provides, among other things, improved and/orengineered non-human animals having humanized genetic material encodinga Lymphocyte-activation gene 3 (Lag-3) polypeptide for determining thetherapeutic efficacy of Lag-3 modulators (e.g., an anti-Lag-3antibodies) for the treatment of cancer, and assays in T cell responsesand signal transduction. It is contemplated that such non-human animalsprovide an improvement in determining the therapeutic efficacy of Lag-3modulators and their potential for Lag-3 blockade. Therefore, thepresent invention is particularly useful for the development ofanti-Lag-3 therapies for the treatment of various cancers and autoimmunediseases, disorders or conditions. In particular, the present inventionencompasses the humanization of a non-human (such as murine) Lag-3 generesulting in expression of a humanized Lag-3 polypeptide on the surfaceof cells of the non-human animal. Such humanized Lag-3 polypeptides havethe capacity to provide a source of human Lag-3⁺ cells for determiningthe efficacy of anti-Lag-3 therapeutics to promote anti-tumor immuneresponses. In some embodiments, non-human animals as described hereindemonstrate augmented immune responses via blockade of Lag-3 signalingthrough the humanized Lag-3 polypeptide expressed on the surface ofcells of the non-human animal. In some embodiments, humanized Lag-3polypeptides comprise a sequence corresponding to an extracellularportion of a human LAG-3 polypeptide, for example, an extracellularportion that contains the first two Ig-like domains of a human LAG-3polypeptide. In some embodiments, humanized Lag-3 polypeptides comprisea sequence corresponding to amino acids 29-260 (or 23-260 or 21-260) ofa human LAG-3 polypeptide. In some embodiments, humanized Lag-3polypeptides comprise a sequence corresponding to the transmembranedomain and/or intracellular tail of a non-human (e.g., rodent such asmurine) Lag-3 polypeptide. In some embodiments, a humanized Lag-3polypeptide comprises an extracellular portion that contains the firsttwo Ig-like domains of a human LAG-3 polypeptide, wherein the remainingportions of the humanized Lag-3 polypeptide are comprised of amino acidsof a non-human (e.g., rodent such as murine) Lag-3 polypeptide. In someembodiments, a humanized Lag-3 polypeptide includes a signal peptidesubstantially identical to the signal peptide of an endogenous non-humanLag-3 polypeptide, an extracellular domain that includes a human portionand a non-human portion wherein the human portion comprises the firsttwo Ig-like domains of a human LAG-3 polypeptide and the non-humanportion comprises the last two Ig-like domains of an endogenousnon-human Lag-3 polypeptide; and the transmembrane and intracellulardomains of an endogenous non-human Lag-3 polypeptide. In someembodiments, non-human animals as described herein comprise a humanizedLag-3 gene that contains genetic material from the non-human animal anda heterologous species (e.g., a human). In some embodiments, non-humananimals as described herein comprise a humanized Lag-3 gene, wherein thehumanized Lag-3 gene comprises exons 2 to 4 of a human LAG-3 gene. Insome certain embodiments, non-human animals as described herein comprisea humanized Lag-3 gene, wherein the humanized Lag-3 gene comprises˜1,741 bp of a human LAG-3 gene corresponding to exons 2 through 4 and aportion of intron 4 (e.g., ˜68 bp) of a human LAG-3 gene. In someembodiments, a non-human animal as described herein comprises ahumanized Lag-3 gene, wherein the humanized Lag-3 gene comprises exon 1of an endogenous Lag-3 gene of the non-human animal, exons 2 to 4 of ahuman LAG-3 gene, and exons 5-8 of an endogenous Lag-3 gene of thenon-human animal, wherein the humanized Lag-3 gene is placed at anendogenous Lag-3 locus and operably linked to the endogenous Lag-3promoter at the locus.

Various aspects of the invention are described in detail in thefollowing sections. The use of sections is not meant to limit theinvention. Each section can apply to any aspect of the invention. Inthis application, the use of “or” means “and/or” unless statedotherwise.

Lymphocyte-Activation Gene 3 (Lag-3)

Lymphocyte-activation gene 3 (Lag-3, also referred to as CD223) is atransmembrane receptor expressed on activated CD4 and CD8 T cells, γδ Tcells, natural killer T cells, B-cells, natural killer cells,plasmacytoid dendritic cells and regulatory T cells. Lag-3 is a memberof the immunoglobulin superfamily, is similar in structure to CD4, andcomprises four extracellular Ig-like domains (also known as domains D1,D2, D3 and D4, see asterisks in the sequence alignment of FIG. 2). Lag-3functions to attenuate the immune response and has been reported to bindmajor histocompatibility complex (MHC) class II molecules (believed tobind through interactions with D1 and D2 domains of Lag-3) and result indelivery of negative signals to Lag-3-expressing cells anddown-regulates antigen-dependent CD4 and CD8 T cell responses.Currently, MHC II is the only known binding partner for Lag-3. Lag-3 hasalso been reported to negatively regulate the ability of T cells toproliferate, produce cytokines and lyse target cells, which is referredto as T cell exhaustion. Further, Lag-3 has been reported to play a rolein enhancing regulatory T (Treg) cell function (Pardoll, D. M., 2012,Nat. Rev. Cancer 12:252-64).

T cell co-stimulatory and co-inhibitory molecules (collectively namedco-signaling molecules) play a crucial role in regulating T cellactivation, subset differentiation, effector function and survival(Chen, L. and D. B. Flies, 2013, Nat. Rev. Immunol. 13:227-42).Following recognition of cognate peptide-MHC complexes onantigen-presenting cells by the T cell receptor, co-signaling receptorsco-localize with T cell receptors at the immune synapse, where theysynergize with TCR signaling to promote or inhibit T cell activation andfunction (Flies, D. B. et al., 2011, Yale J. Biol. Med. 84:409-21). Theultimate immune response is regulated by a balance betweenco-stimulatory and co-inhibitory signals, which have been referred to as“immune checkpoints” (Pardoll, D. M., supra). Such “immune checkpoints”can be described as molecules operating in the immune system to eitherturn up or down signals, especially T cell signals. Lag-3 functions asone of many “immune checkpoint” in mediating peripheral T celltolerance.

A more thorough and detailed understanding of Lag-3-mediated functionsand the Lag-3 pathway in tumor, auto- and infectious immunity is neededto develop practical targeted therapies for future treatment of humanpatients.

Lag-3 Sequences

Exemplary rodent (e.g., rat and mouse), human, and humanizedlymphocyte-activation gene 3 (Lag-3) sequences are set forth in FIG. 8.An exemplary synthetic DNA fragment for humanization of a non-humanLag-3 gene is also set forth in FIG. 8. For mRNA sequences, bold fontindicates coding sequence, and consecutive exons, where indicated, areseparated by alternating underlined text; for humanized mRNA sequences,human sequences are contained within parentheses. For amino acidsequences, transmembrane sequences are indicated by underlined font; forhumanized amino acid sequences, human sequences are indicated in boldfont and contained within parentheses.

DNA Constructs

Typically, a polynucleotide molecule containing a Lag-3 gene, in wholeor in part, is inserted into a vector, preferably a DNA vector, in orderto replicate the polynucleotide molecule in a suitable host cell.

Depending on size, a Lag-3 gene or Lag-3-encoding sequence can be cloneddirectly from cDNA sources available from commercial suppliers ordesigned in silico based on published sequences available from GenBank.Alternatively, bacterial artificial chromosome (BAC) libraries canprovide heterologous Lag-3 sequences from genes of interest (e.g., aheterologous Lag-3 gene). BAC libraries contain an average insert sizeof 100-150 kb and are capable of harboring inserts as large as 300 kb(Shizuya, H. et al., 1992, Proc. Natl. Acad. Sci., U.S.A. 89:8794-7;Swiatek, P. J. and T. Gridley, 1993, Genes Dev. 7:2071-84; Kim, U. J. etal., 1996, Genomics 34:213-8; herein incorporated by reference). Forexample, human and mouse genomic BAC libraries have been constructed andare commercially available (e.g., Invitrogen, Carlsbad Calif.). GenomicBAC libraries can also serve as a source of heterologous Lag-3 sequencesas well as transcriptional control regions.

Alternatively, heterologous Lag-3 sequences may be isolated, clonedand/or transferred from yeast artificial chromosomes (YACs). An entireheterologous gene or locus can be cloned and contained within one or afew YACs. If multiple YACs are employed and contain regions ofoverlapping homology, they can be recombined within yeast host strainsto produce a single construct representing the entire locus. YAC armscan be additionally modified with mammalian selection cassettes byretrofitting to assist in introducing the constructs into embryonicstems cells or embryos by methods known in the art and/or describedherein.

Exemplary mRNA and amino acid sequences for use in constructing ahumanized Lag-3 gene in a non-human animal are provided herein, e.g., inFIG. 8. Other heterologous Lag-3 sequences can also be found in theGenBank database or other sequence databases known in the art.

DNA constructs containing Lag-3 sequences as described herein, in someembodiments, comprise human LAG-3 genomic sequences encoding anextracellular portion of a human LAG-3 polypeptide, e.g., at least aminoacids 29-260 (or 23-260 or 21-260) of a human LAG-3 polypeptide,operably linked to non-human regulatory sequences (e.g., a rodentpromoter) for expression in a transgenic non-human animal. In someembodiments, DNA constructs containing Lag-3 sequences as describedherein comprise human LAG-3 genomic sequences encoding at least aminoacids 29-260 of a human LAG-3 polypeptide operably linked to a non-humanLag-3 promoter and one or more non-human Lag-3 exons (e.g., endogenousLag-3 exons). Human and/or non-human Lag-3 sequences included in DNAconstructs described herein may be identical or substantially identicalwith human and/or non-human Lag-3 sequences found in nature (e.g.,genomic), artificial (e.g., synthetic) or may be engineered by the handof man. In some embodiments, Lag-3 sequences are synthetic in origin,and include a sequence or sequences that are found in a human LAG-3 genefound in nature. For example, a DNA construct can include synthetic DNAthat corresponds to exons 2 through 4 of a human LAG-3 gene, and thatencodes an extracellular portion of a human LAG-3 polypeptide, e.g., atleast amino acids 29-260 of a human LAG-3 polypeptide, operably linkedto non-human Lag-3 regulatory (e.g., promoter) and coding sequences(e.g., one or more non-human exons) so that a Lag-3 polypeptide havinghuman and non-human portions is encoded by the resulting DNA construct.In some embodiments, Lag-3 sequences comprise a sequence naturallyassociated with a heterologous Lag-3 gene (i.e., a human LAG-3 gene). Insome embodiments, Lag-3 sequences comprise a sequence that is notnaturally associated with a heterologous Lag-3 gene (i.e., a human LAG-3gene). In some embodiments, Lag-3 sequences comprise a sequence that isoptimized for expression in a non-human animal. In some embodiments,heterologous Lag-3 sequences operably linked to non-human Lag-3sequences each encode a portion of a Lag-3 polypeptide that appears inseparate polypeptides in nature. If additional sequences are useful inoptimizing expression of heterologous Lag-3 sequences, such sequencescan be cloned using existing sequences as probes. Additional sequencesnecessary for maximizing expression of a heterologous Lag-3 gene orheterologous Lag-3-encoding sequence can be obtained from genomicsequences or other sources depending on the desired outcome.

DNA constructs can be prepared using methods known in the art. Forexample, a DNA construct can be prepared as part of a larger plasmid.Such preparation allows the cloning and selection of the correctconstructions in an efficient manner as is known in the art. DNAfragments containing one or more nucleotide coding sequences asdescribed herein can be located between convenient restriction sites onthe plasmid so that they can be easily isolated from the remainingplasmid sequences for incorporation into the desired animal.

Various methods employed in preparation of plasmids and host organismscontaining them are known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning: A Laboratory Manual, 2ndEd., ed. by Sambrook, J. et al., Cold Spring Harbor Laboratory Press:1989.

Production of Non Human Animals Having A Humanized Lymphocyte-ActivationGene 3

Non-human animals are provided that express humanized Lag-3 polypeptideson the surface of cells of the non-human animals resulting from agenetic modification of an endogenous locus (e.g., a Lag-3 locus) of thenon-human animal that encodes a Lag-3 polypeptide. Suitable examplesdescribed herein include rodents, in particular, mice.

A humanized Lag-3 gene, in some embodiments, comprises genetic materialfrom a heterologous species (e.g., humans), wherein the humanized Lag-3gene encodes a Lag-3 polypeptide that comprises the encoded portion ofthe genetic material from the heterologous species. In some embodiments,a humanized Lag-3 gene as described herein comprises genomic DNA of aheterologous species that encodes an extracellular portion of a Lag-3polypeptide that is expressed on the plasma membrane of a cell.Non-human animals, embryos, cells and targeting constructs for makingnon-human animals, non-human embryos, and cells containing saidhumanized Lag-3 gene are also provided.

In some embodiments, an endogenous Lag-3 gene is deleted. In someembodiments, an endogenous Lag-3 gene is altered, wherein a portion ofthe endogenous Lag-3 gene is replaced with a heterologous sequence(e.g., a human LAG-3 sequence, in whole or in part). In someembodiments, all or substantially all of an endogenous Lag-3 gene isreplaced with a heterologous gene (e.g., a human LAG-3 gene). In someembodiments, a portion of a heterologous Lag-3 gene is inserted into anendogenous non-human Lag-3 gene at an endogenous Lag-3 locus. In someembodiments, the heterologous gene is a human gene. In some embodiments,the modification or humanization is made to one of the two copies of theendogenous Lag-3 gene, giving rise to a non-human animal that isheterozygous with respect to the humanized Lag-3 gene. In otherembodiments, a non-human animal is provided that is homozygous for ahumanized Lag-3 gene.

In various aspects, a non-human animal contains a human LAG-3 gene, inwhole or in part, at an endogenous non-human Lag-3 locus. Thus, suchnon-human animals can be described as having a heterologous Lag-3 gene.The replaced, inserted, modified or altered Lag-3 gene at the endogenousLag-3 locus or a polypeptide expressed from such gene can be detectedusing a variety of methods including, for example, PCR, Western blot,Southern blot, restriction fragment length polymorphism (RFLP), or again or loss of allele assay. In some embodiments, the non-human animalis heterozygous with respect to the humanized Lag-3 gene.

In various embodiments, a humanized Lag-3 gene as described hereinincludes exons 2 through 4 of a human LAG-3 gene.

In various embodiments, a humanized Lag-3 gene as described hereinincludes a Lag-3 gene that has a second, third and fourth exon eachhaving a sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identicalto a second, third and fourth exon that appear in SEQ ID NO:5.

In various embodiments, a humanized Lag-3 gene as described hereinincludes a Lag-3 gene that has a second, third and fourth exon eachhaving a sequence that is substantially identical or identical to asecond, third and fourth exon that appear in SEQ ID NO:5.

In various embodiments, a humanized Lag-3 gene as described hereincomprises a sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identical to SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11.

In various embodiments, a humanized Lag-3 gene as described hereincomprises a sequence that is substantially identical or identical to SEQID NO:9, SEQ ID NO:10 or SEQ ID NO:11.

In various embodiments, a humanized Lag-3 gene as described herein is orcomprises SEQ ID NO:10.

In various embodiments, a humanized Lag-3 gene as described herein is orcomprises SEQ ID NO:11.

In various embodiments, a humanized Lag-3 gene as described hereincomprises exons 1, 5, 6, 7 and 8 of a non-human Lag-3 gene, for example,an endogenous Lag-3 gene of a non-human animal.

In various embodiments, a humanized Lag-3 gene as described hereincomprises a first, fifth, sixth, seventh and eighth exon each having asequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to afirst, fifth, sixth, seventh and eighth exon that appear in SEQ ID NO:1or SEQ ID NO:3.

In various embodiments, a humanized Lag-3 gene as described hereincomprises a first, fifth, sixth, seventh and eighth exon each having asequence that is substantially identical or identical to a first, fifth,sixth, seventh and eighth exon that appear in SEQ ID NO:1 or SEQ IDNO:3.

In various embodiments, a humanized Lag-3 gene as described hereincomprises a 5′ untranslated region and a 3′ untranslated region of anon-human Lag-3 gene, for example, an endogenous Lag-3 gene of anon-human animal.

In various embodiments, a humanized Lag-3 gene as described hereincomprises a 5′ untranslated region and a 3′ untranslated region eachhaving a sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identicalto a 5′ untranslated region and a 3′ untranslated region that appear inSEQ ID NO:1 or SEQ ID NO:3.

In various embodiments, a humanized Lag-3 gene as described hereincomprises a 5′ untranslated region and a 3′ untranslated region eachhaving a sequence that is substantially identical or identical to a 5′untranslated region and a 3′ untranslated region that appear in SEQ IDNO:1 or SEQ ID NO:3.

In various embodiments, a humanized Lag-3 gene as described hereincomprises a nucleotide coding sequence (e.g., a cDNA sequence) at least50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more) identical to a nucleotide codingsequence that appears in SEQ ID NO:7.

In various embodiments, a humanized Lag-3 gene as described hereincomprises a nucleotide coding sequence (e.g., a cDNA sequence) that issubstantially identical or identical to a nucleotide coding sequencethat appears in SEQ ID NO:7.

In various embodiments, a humanized Lag-3 gene as described hereinencodes a Lag-3 polypeptide having an amino acid sequence at least 50%(e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more) identical to an amino acid sequencethat appears in SEQ ID NO:8.

In various embodiments, a humanized Lag-3 gene as described hereinencodes a Lag-3 polypeptide having an amino acid sequence that issubstantially identical or identical to an amino acid sequence thatappears in SEQ ID NO:8.

In various embodiments, a humanized Lag-3 polypeptide produced by anon-human animal as described herein has an extracellular portion, whichextracellular portion comprises an amino acid sequence at least 50%(e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues 29-260of SEQ ID NO:6 or SEQ ID NO:8.

In various embodiments, a humanized Lag-3 polypeptide produced by anon-human animal as described herein has an extracellular portionsubstantially identical to an extracellular portion (e.g., anextracellular portion that contains the first two Ig-like domains) of ahuman LAG-3 polypeptide. In some embodiments, an extracellular portionof a human LAG-3 polypeptide is represented by amino residues 21-260,23-260, or 29-260 of a human LAG-3 polypeptide, such as a human LAG-3polypeptide as set forth in SEQ ID NO: 6.

In various embodiments, a humanized Lag-3 polypeptide produced by anon-human animal as described herein has an extracellular portion, whichextracellular portion comprises an amino acid sequence that issubstantially identical or identical to amino acid residues 29-260 ofSEQ ID NO:6 or SEQ ID NO:8.

In various embodiments, a humanized Lag-3 polypeptide produced by anon-human animal as described herein has an extracellular portion, whichextracellular portion comprises an amino acid sequence at least 50%(e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues 23-260of SEQ ID NO:6 or SEQ ID NO:8.

In various embodiments, a humanized Lag-3 polypeptide produced by anon-human animal as described herein has an extracellular portion, whichextracellular portion comprises an amino acid sequence that issubstantially identical or identical to amino acid residues 23-260 ofSEQ ID NO:6 or SEQ ID NO:8.

In various embodiments, a humanized Lag-3 polypeptide produced by anon-human animal as described herein has an extracellular portion, whichextracellular portion comprises an amino acid sequence at least 50%(e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more) identical to amino acid residues 21-260of SEQ ID NO:6 or SEQ ID NO:8.

In various embodiments, a humanized Lag-3 polypeptide produced by anon-human animal as described herein has an extracellular portion, whichextracellular portion comprises an amino acid sequence that issubstantially identical or identical to amino acid residues 21-260 ofSEQ ID NO:6 or SEQ ID NO:8.

In various embodiments, a humanized Lag-3 polypeptide produced by anon-human animal as described herein has a transmembrane portion and acytoplasmic portion of a non-human Lag-3 polypeptide, for example, thetransmembrane and cytoplasmic domains of an endogenous Lag-3 polypeptideof a non-human animal. In some embodiments, the transmembrane andcytoplasmic domain sequences of a non-human Lag-3 polypeptide are thoseillustrated in FIG. 8. In some embodiments, a humanized Lag-3polypeptide produced by a non-human animal as described herein also hasan extracellular portion containing the last two Ig-liked domains of anon-human Lag-3 polypeptide.

In various embodiments, a humanized Lag-3 polypeptide produced by anon-human animal as described herein has an amino acid sequence at least50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more) identical to an amino acidsequence of SEQ ID NO:8.

In various embodiments, a humanized Lag-3 polypeptide produced by anon-human animal as described herein has an amino acid sequence that issubstantially identical or identical to an amino acid sequence of SEQ IDNO:8.

Compositions and methods for making non-human animals that express ahumanized Lag-3 polypeptide, including specific polymorphic forms,allelic variants (e.g., single amino acid differences) or alternativelyspliced isoforms, are provided, including compositions and methods formaking non-human animals that express such polypeptides from a humanpromoter and a human regulatory sequence. In some embodiments,compositions and methods for making non-human animals that express suchproteins from a non-human promoter and a non-human regulatory sequenceare also provided. In some embodiments, compositions and methods formaking non-human animals that express such proteins from an endogenouspromoter and an endogenous regulatory sequence are also provided. Insome certain embodiments, endogenous promoters and endogenous regulatorysequences are endogenous rodent promoters and endogenous rodentregulatory sequences. The methods include inserting the genetic materialencoding a human LAG-3 polypeptide, in whole or in part, at a preciselocation in the genome of a non-human animal that corresponds to anendogenous Lag-3 gene thereby creating a humanized Lag-3 gene thatexpresses a Lag-3 protein that is human in whole or in part. In someembodiments, the methods include inserting genomic DNA corresponding toexons 2, 3 and 4 of a human LAG-3 gene into an endogenous Lag-3 gene ofthe non-human animal thereby creating a humanized gene that encodes aLag-3 polypeptide that contains a human portion containing amino acidsencoded by the inserted exons.

Where appropriate, the coding region of the genetic material orpolynucleotide sequence(s) encoding a human (or humanized) Lag-3polypeptide in whole or in part may be modified to include codons thatare optimized for expression from cells in the non-human animal (e.g.,see U.S. Pat. Nos. 5,670,356 and 5,874,304). Codon optimized sequencesare synthetic sequences, and preferably encode the identical polypeptide(or a biologically active fragment of a full length polypeptide whichhas substantially the same activity as the full length polypeptide)encoded by the non-codon optimized parent polynucleotide. In someembodiments, the coding region of the genetic material encoding a human(or humanized) Lag-3 polypeptide, in whole or in part, may include analtered sequence to optimize codon usage for a particular cell type(e.g., a rodent cell). For example, the codons of the genomic DNAcorresponding to exons 2, 3 and 4 of a human LAG-3 gene to be insertedinto an endogenous Lag-3 gene of a non-human animal (e.g., a rodent) maybe optimized for expression in a cell of the non-human animal. Such asequence may be described as a codon-optimized sequence.

Methods for generating transgenic non-human animals, including knockoutsand knock-ins, are well known in the art (see, e.g., Gene Targeting: APractical Approach, Joyner, ed., Oxford University Press, Inc. (2000)).For example, generation of transgenic rodents may optionally involvedisruption of the genetic loci of one or more endogenous rodent genes(or gene segments) and introduction of one or more heterologous genes(or Lag-3 encoding sequences) into the rodent genome, in someembodiments, at the same location as an endogenous rodent gene (or genesegments).

In some embodiments, heterologous (or humanized) Lag-3 genes orheterologous Lag-3-encoding sequences as described herein are introducedrandomly in the genome of a rodent. In such embodiments, rodentscomprising, containing or otherwise harboring randomly introducedheterologous (or humanized Lag-3 genes or heterologous Lag-3-encodingsequences can be characterized as having a heterologous Lag-3 transgeneor heterologous Lag-3 transgene construct. Typically, a transgene and/ortransgene construct includes, among other things, a nucleic acidsequence (encoding e.g., a polypeptide of interest, in whole or in part)that is introduced into a non-human cell (e.g., a rodent embryonic stemcell) by the hand of man using methods described herein or otherwiseknown in the art. Further, a transgene may be partly or entirelyheterologous, i.e., foreign, to a non-human animal or cell into which itis introduced. A transgene can further include one or moretranscriptional regulatory sequences and any other nucleic acid, such asintrons or promoters, which may be necessary for expression of aselected nucleic acid sequence. In some embodiments, heterologous (orhumanized) Lag-3 genes or heterologous Lag-3-encoding sequences asdescribed herein are introduced into an endogenous Lag-3 gene in thegenome of a rodent; in some certain embodiments, an endogenous Lag-3gene locus is altered, modified, or engineered to contain human Lag-3sequences (or gene fragments) operably linked to one or more non-humanLag-3 sequences (or gene fragments).

A humanized Lag-3 gene approach employs a relatively minimalmodification of the endogenous protein interactions and signaling andresults in natural Lag-3-mediated signal transduction in the non-humananimal, in various embodiments, because the genomic sequence of theLag-3 sequences are modified in a single fragment and therefore retainnormal functionality by including necessary regulatory sequences. Thus,in such embodiments, the Lag-3 gene modification does not affect othersurrounding genes or other endogenous Lag-3-interacting genes (e.g., MHCclass II molecules). Further, in various embodiments, the modificationdoes not affect the assembly of a functional Lag-3 transmembranepolypeptide on the cell membrane and maintains normal effector functionsvia binding and subsequent signal transduction through the cytoplasmicportion of the polypeptide which is unaffected by the modification.

A schematic illustration (not to scale) of the genomic organization ofan endogenous murine Lag-3 gene and a human LAG-3 gene is provided inFIG. 1. An exemplary method for humanizing an endogenous murine Lag-3gene using a genomic fragment containing exons 2, 3 and 4 and a portionof intron 4 (e.g., about 68 bp) of a human LAG-3 gene is provided inFIG. 3. As illustrated, a 1,741 bp synthetic DNA fragment correspondingto exons 2, 3 and 4 and a portion of intron 4 of a human LAG-3 gene isinserted into the place of a 1,750 bp sequence of an endogenous murineLag-3 gene locus by a targeting construct. The 1,741 bp synthetic DNAfragment may be cloned directly from human DNA or synthesized from asource sequence (e.g., GenBank accession no. NM_002286.5, SEQ ID NO:9).This genomic DNA includes the portion of the gene that encodes at leastamino acid residues 29-260 (or 23-260 or 21-260) of a human LAG-3polypeptide responsible for ligand binding.

A non-human animal (e.g., a mouse) having a humanized Lag-3 gene at theendogenous Lag-3 locus can be made by any method known in the art. Forexample, a targeting vector can be made that introduces a human LAG-3gene in whole or in part with a selectable marker gene. FIG. 3illustrates a targeting vector that contains an endogenous Lag-3 locusof a mouse genome comprising an insertion of a 1,741 bp synthetic DNAfragment that corresponds to exons 2-4 and the first 68 bp of intron 4of a human LAG-3 gene. As illustrated, the targeting construct containsa 5′ homology arm containing sequence upstream of exon 2 (i.e., exon 1,etc.) of an endogenous murine Lag-3 gene (˜46 Kb), followed by the 1,741bp synthetic DNA fragment, a drug selection cassette (e.g., a neomycinresistance gene flanked on both sides by loxP sequences; ˜5 Kb), and a3′ homology arm containing the remaining sequence of an endogenousmurine exons 5-8 of an endogenous murine Lag-3 gene (˜53 Kb). Thetargeting construct contains a self-deleting drug selection cassette(e.g., a neomycin resistance gene flanked by loxP sequences; see U.S.Pat. Nos. 8,697,851, 8,518,392 and 8,354,389, all of which areincorporated herein by reference). Upon electroporation in embryonicstem cells, a modified endogenous Lag-3 gene is created that includes1,741 bp of a human LAG-3 gene (i.e., exons 2-4 and the first 68 bp ofintron 4) in the place of 1,750 bp of an endogenous wild type Lag-3gene, which is contained in the targeting vector. A humanized Lag-3 geneis created resulting in a cell or non-human animal that expresses ahumanized Lag-3 polypeptide that contains amino acids encoded by the1,741 bp synthetic DNA fragment (i.e., exons 2-4 and the first 68 bp ofintron 4 of a human LAG-3 gene). The drug selection cassette is removedin a development-dependent manner, i.e., progeny derived from mice whosegerm line cells containing the humanized Lag-3 gene described above willshed the selectable marker from differentiated cells during development(see bottom of FIG. 3).

In some embodiments, a non-human animal having a humanized Lag-3 gene asdescribed herein can be characterized as transgenic for the humanizedLag-3 gene or a transgenic Lag-3 non-human animal. Such descriptions areused interchangeably herein and refer to any non-naturally occurringnon-human animal in which one or more of the cells of the non-humananimal contain a heterologous Lag-3 nucleic acid sequence and/orLag-3-encoding sequence, in whole or in part, as described herein. Insome embodiments, a heterologous Lag-3 nucleic acid sequence and/orLag-3-encoding sequence, in whole or in part, is introduced into a cell,directly or indirectly by introduction into a precursor cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. In such embodiments, geneticmanipulation does not include classic breeding techniques, but rather isdirected to introduction of recombinant DNA molecule(s) that contain aheterologous Lag-3 nucleic acid sequence and/or Lag-3-encoding sequence,in whole or in part, as described herein. Such a molecule may beintegrated within a chromosome, or it may be extrachromosomallyreplicating DNA. As described herein, transgenic non-human animalsincludes animals that are heterozygous or homozygous for a heterologousLag-3 nucleic acid sequence and/or Lag-3-encoding sequence, in whole orin part, and/or animals that have single or multiple copies of aheterologous Lag-3 nucleic acid sequence and/or Lag-3-encoding sequence,in whole or in part, as described herein.

A transgenic founder non-human animal can be identified based upon thepresence of a humanized Lag-3 gene in its genome and/or expression ofLag-3 polypeptides containing amino acids encoded by the insertedgenetic material in tissues or cells of the non-human animal. Atransgenic founder non-human animal can then be used to breed additionalnon-human animals carrying the humanized Lag-3 gene thereby creating aseries of non-human animals each carrying one or more copies of ahumanized Lag-3 gene. Moreover, transgenic non-human animals carrying ahumanized Lag-3 gene can further be bred to other transgenic non-humananimals carrying other transgenes (e.g., human immunoglobulin genes) asdesired.

Transgenic non-human animals may also be produced to contain selectedsystems that allow for regulated or directed expression of the humanizedLag-3 gene (or humanized Lag-3 transgene). Exemplary systems include theCre/loxP recombinase system of bacteriophage P1 (see, e.g., Lakso, M. etal., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:6232-6236) and the FLP/Frtrecombinase system of S. cerevisiae (O'Gorman, S. et al, 1991, Science251:1351-1355). Such animals can be provided through the construction of“double” transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene comprising a selected modification (e.g., ahumanized Lag-3 gene or transgene) and the other containing a transgeneencoding a recombinase (e.g., a Cre recombinase).

The non-human animals as described herein may be prepared as describedabove, or using methods known in the art, to comprise additional humanor humanized genes, oftentimes depending on the intended use of thenon-human animal. Genetic material of such additional human or humanizedgenes may be introduced through the further alteration of the genome ofcells (e.g., embryonic stem cells) having the genetic modifications asdescribed above or through breeding techniques known in the art withother genetically modified strains as desired. In some embodiments,non-human animals as described herein are prepared to further compriseone or more human or humanized genes selected from Programmed cell deathprotein 1 (PD-1), Programmed death-ligand 1 (PD-L1), and cytotoxicT-lymphocyte-associated protein 4 (CTLA-4). In some embodiments,non-human animals as described herein may be prepared by introducing atargeting vector, as described herein, into a cell from a modifiedstrain. In some embodiments, non-human animals as described herein areprepared to further comprise a human or humanized Programmed cell deathprotein 1 (Pdcd1) gene. In some embodiments, non-human animals asdescribed herein comprise a humanized Lag-3 gene, as described herein,and genetic material from a heterologous species (e.g., humans), whereinthe genetic material encodes, in whole or in part, one or moreheterologous proteins selected from Programmed cell death protein 1(PD-1), Programmed death-ligand 1 (PD-L1), and cytotoxicT-lymphocyte-associated protein 4 (CTLA-4). In some certain embodiments,non-human animals as described herein comprise a humanized Lag-3 gene asdescribed herein and genetic material from a heterologous species (e.g.,humans), wherein the genetic material encodes, in whole or in part, aheterologous (e.g., human) PD-1 polypeptide. In some certainembodiments, non-human animals as described herein further comprise aPdcd1 gene that comprises an endogenous portion and a human portion(e.g., exon 2 and exon 3, in whole or in part, of a human PDCD1 gene),wherein the human portion encodes substantially all of the extracellulardomain of a human PD-1 polypeptide (e.g., amino acids corresponding toresidues 27-169 or 26-169 of a human PD-1 polypeptide) and theendogenous portion encodes the intracellular domain of an endogenousPD-1 polypeptide; in some embodiments, the human portion and theendogenous portion are operably linked to an endogenous Pdcd1 promoter.In some certain embodiments, non-human animals as described hereinfurther comprise a Pdcd1 gene that includes genetic material thatencodes substantially all of the extracellular domain of a human PD-1polypeptide (e.g., genetic material that encodes amino acidscorresponding to residues 27-169 or 26-169 of a human PD-1 polypeptide;see, e.g., SEQ ID NO:23 of PCT/US15/36649, filed 19 Jun. 2015 andpublished as WO2015196051, and/or SEQ ID NO:23 of U.S. patentapplication Ser. No. 14/744,592, filed 19 Jun. 2015 and published as US2015-0366174 A1; incorporated herein by reference). GenBank accessionnos. NM_005018.2 and NP_005009.2, and UniProt ID Q15116 providerepresentative source sequences of a human PDCD1 gene and human PD-1polypeptide from which a desired human portion may be obtained.

For example, as described herein, non-human animals comprising ahumanized Lag-3 gene as described herein may further comprise (e.g., viacross-breeding or multiple gene targeting strategies) one or moremodifications as described in PCT/US 15/36649, filed 19 Jun. 2015 andpublished as 102015196051, and U.S. patent application Ser. No.14/744,592, filed 19 Jun. 2015 and published as US 2015-0366174 A1;these applications are incorporated herein by reference in theirentirety. In certain embodiments, a rodent comprising a humanized Lag-3gene as described herein is crossed to a rodent comprising a humanizedPdcd1 gene (e.g., exon 2 and a portion of exon 3 of a human PDCD1 geneoperably linked to exons 1, a portion of exon 3, 4 and 5 of anendogenous rodent Pdcd1 gene so that the humanized Pdcd1 gene encodes aPD-1 polypeptide includes an extracellular portion from a human PD-1polypeptide (e.g., corresponding to amino acids residues 27-169 or26-169) and an intracellular portion from a rodent PD-1 polypeptideprotein (see, e.g., SEQ ID NOs:5 and 6 of PCT/US15/36649, filed 19 Jun.2015 and published as WO2015196051, and/or SEQ ID NOs:5 and 6 of U.S.patent application Ser. No. 14/744,592, filed 19 Jun. 2015 and publishedas US 2015-0366174 A1; incorporated herein by reference). In certainembodiments, a rodent comprising a humanized Lag-3 gene as describedherein is crossed to a rodent comprising a humanized Pdcd1 gene, whichhumanized Pdcd1 gene includes genetic material that encodesextracellular domain of a human PD-1 polypeptide (e.g., genetic materialthat encodes amino acids corresponding to residues 27-169 or 26-169 of ahuman PD-1 polypeptide; see, e.g., SEQ ID NO:23 of PCT/US15/36649, filed19 Jun. 2015 and published as WO2015196051, and/or SEQ ID NO:23 of U.S.patent application Ser. No. 14/744,592, filed 19 Jun. 2015 published asUS 2015-0366174 A1; incorporated herein by reference).

In some embodiments, a humanized Pdcd1 gene comprises a non-human (e.g.,rodent) Pdcd1 exon 1, a human PDCD1 exon 2, an exon 3 (which comprises aportion of exon 3 of a human PDCD1 gene and a portion of exon 3 of anon-human (e.g., rodent) Pdcd1 gene), and exons 4-5 of a non-human(e.g., rodent) Pdcd1 gene, and wherein in some embodiments, the portionof exon 3 of a human PDCD1 gene is the 5′ portion of human exon 3 thatencodes amino acids as part of the extracellular domain of a human PD-1polypeptide, such as amino acids of a PD-1 stalk sequence, and theportion of exon 3 of a non-human Pdcd1 gene is the 3′ portion ofnon-human exon 3 that encodes amino acids as part of the transmembranedomain of a non-human PD-1 polypeptide. In specific embodiments, ahumanized Pdcd1 gene encodes a humanized PD-1 polypeptide that includesan extracellular domain substantially identical to the extracellulardomain of a human PD-1 polypeptide, a transmembrane domain substantiallyidentical to the transmembrane domain of a rodent PD-1 polypeptide, andthe intracellular domain of a rodent PD-1 polypeptide.

Although embodiments employing a humanized Lag-3 gene in a mouse (i.e.,a mouse with a Lag-3 gene that encodes a Lag-3 polypeptide that includesa human portion and a mouse portion) are extensively discussed herein,other non-human animals that comprise a humanized Lag-3 gene are alsoprovided. In some embodiments, such non-human animals comprise ahumanized Lag-3 gene operably linked to a rodent Lag-3 promoter. In someembodiments, such non-human animals comprise a humanized Lag-3 geneoperably linked to an endogenous Lag-3 promoter; in some embodiments, anendogenous rodent Lag-3 promoter. In some embodiments, such non-humananimals express a humanized Lag-3 polypeptide from an endogenous locus,wherein the humanized Lag-3 polypeptide comprises at least amino acidresidues 29-260 (e.g., 29-260, 23-260 or 21-260) of a human LAG-3polypeptide. Such non-human animals include any of those which can begenetically modified to express a Lag-3 polypeptide as disclosed herein,including, e.g., mammals, e.g., mouse, rat, rabbit, pig, bovine (e.g.,cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret,primate (e.g., marmoset, rhesus monkey), etc. For example, for thosenon-human animals for which suitable genetically modifiable ES cells arenot readily available, other methods are employed to make a non-humananimal comprising the genetic modification. Such methods include, e.g.,modifying a non-ES cell genome (e.g., a fibroblast or an inducedpluripotent cell) and employing somatic cell nuclear transfer (SCNT) totransfer the genetically modified genome to a suitable cell, e.g., anenucleated oocyte, and gestating the modified cell (e.g., the modifiedoocyte) in a non-human animal under suitable conditions to form anembryo.

Methods for modifying a non-human animal genome (e.g., a pig, cow,rodent, chicken, etc. genome) include, e.g., employing a zinc fingernuclease (ZFN), a transcription activator-like effector nuclease(TALEN), or a Cas protein (i.e., a CRISPR/Cas system) to modify a genometo include a humanized Lag-3 and/or humanized Pdcd1 gene.

Methods Employing Non-Human Animals Having Humanized Lag-3 Genes

The use of humanized mice for biomedical research has advancedunderstanding of various aspects of the function of human cells, inparticular, human immune cells. Indeed, the use of variousimmunocompromised strains has provided valuable in vivo systems for theinvestigation of human immune cells. However, they are not withoutlimitation. For example, the use of these models has highlighteddistinct differences in mammalian biology, in particular, mammalianimmunology. The use of immunocompromised mice for evaluating targetimmunomodulation is not ideal as aberrant immune function observed insuch mice often complicates understanding of displayed phenotypes. Thisis especially true in the context of the immune response to tumors.Until recently, investigation of the immune responses to tumors (e.g., Tcell responses) in mice was confounded by, among other things,mismatching of HLA due to different sources (i.e., donors) of engraftedhuman cells (Shultz, L. D. et al., 2010, Proc. Nat. Acad. Sci. U.S.A.107(29):13022-7).

Tumor cells can escape immune recognition and effector response byaltering co-stimulatory pathways in T cells. Co-stimulatory pathwaysmediate the balance among T cell activation, tolerance, andimmune-mediated tissue damage by regulating the magnitude and durationof an immune response within the environment of antigenic stimulation.Lymphocyte-activation gene 3 (Lag-3) is a negative regulator of T cellactivity and controls the size of the memory T cell pool (Workman, C. J.et al., 2004, J. Immunol. 172:5450-5). The functional role of Lag-3 onantigen-experienced CD4⁺ and CD8⁺ T cells has been reported, andupregulation of Lag-3 on these cells following T cell receptor (TCR)engagement leads to negative effects on T cell proliferation,activation, and proinflammatory cytokine production (Huard, B. et al.,1994, Eur. J. Immunol. 24(12):3216-21; Huard, B. et al., 1994,Immunogenetics 39(3):213-7; Macon-Lemaitre, L. and F. Triebel, 2005,Immunol. 115:170-8; Goldberg, M. V. and C. G. Drake, 2011, Curr. Top.Microbiol. Immunol. 344:269-78). Lag-3 blockade has been reported toenhance anti-tumor response in T cells (Grosso, J. F. et al., 2007, J.Clin. Invest. 117:3383-92). Other inhibitory receptors involved innegative regulation of T cell activity include programmed cell deathprotein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4(CTLA-4). Along with its ligands Programmed Death Ligand 1 (PD-L1, B7-H1or CD274) and PD-L2 (B7-DC or CD273), PD-1 delivers a checkpoint signalcritical for the establishment and maintenance of immune tolerance toenvironmental and self-antigens (Francisco, L. M. et al., 2010, Immunol.Rev. 236:219-42). PD-1, like Lag-3, has been reported to play a role inevading anti-tumor immunity by allowing tumor cells to escape immunesurveillance by the host immune system (Zou, C. 2008, Nat. Rev. Immunol.8:467-77).

The present invention is, among other things, based on the recognitionthat the expression of these inhibitory receptors is associated withcompromised antigen-specific T cell function in the context of cancer.The present invention is also based on the recognition that the creationof an in vivo system that exploits immune checkpoints can be made usinga humanized Lag-3 gene and/or a humanized Pdcd1 gene as describedherein. Such an improved in vivo system allows for the development ofanti-immune checkpoint therapeutics and/or therapeutic regimens thatfocus on stimulating anti-tumor immunity in cancer patients. Further,such an improved in vivo system also provides for the development oftherapeutics and/or therapeutic regimens that focus on augmenting T cellactivity in infectious and/or autoimmune diseases.

Non-human animals as described herein provide an improved in vivo systemand source of biological materials (e.g., cells) expressing human (orhumanized) Lag-3 that are useful for a variety of assays. In variousembodiments, non-human animals as described herein are used to developtherapeutics that target Lag-3 and/or modulate Lag-3 signaling (e.g.,disrupting interactions with Lag-3 binding partners, such as MHC classII molecules). In various embodiments, non-human animals as describedherein are used to screen and develop candidate therapeutics (e.g.,antibodies) that block interaction of human Lag-3 with human MHC classII molecules. In various embodiments, non-human animals as describedherein are used to determine the binding profile of antagonists and/oragonists of a humanized Lag-3 on the surface of a cell of a non-humananimal as described herein; in some embodiments, non-human animals asdescribed herein are used to determine the epitope or epitopes of one ormore candidate therapeutic antibodies that bind human Lag-3.

In various embodiments, non-human animals as described herein are usedto determine the pharmacokinetic profiles of anti-Lag-3 antibodies. Invarious embodiments, one or more non-human animals as described hereinand one or more control or reference non-human animals are each exposedto one or more candidate therapeutic anti-Lag-3 antibodies at variousdoses (e.g., 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 1mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/mg, 7.5 mg/kg, 10 mg/kg, 15mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg or more).Candidate therapeutic antibodies may be dosed via any desired route ofadministration including parenteral and non-parenteral routes ofadministration. Parenteral routes include, e.g., intravenous,intraarterial, intraportal, intramuscular, subcutaneous,intraperitoneal, intraspinal, intrathecal, intracerebroventricular,intracranial, intrapleural or other routes of injection. Non-parenteralroutes include, e.g., oral, nasal, transdermal, pulmonary, rectal,buccal, vaginal, ocular. Administration may also be by continuousinfusion, local administration, sustained release from implants (gels,membranes or the like), and/or intravenous injection. Blood is isolatedfrom non-human animals (humanized and control) at various time points(e.g., 0 hr, 6 hr, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, or up to 30 or more days).Various assays may be performed to determine the pharmacokineticprofiles of administered candidate therapeutic antibodies using samplesobtained from non-human animals as described herein including, but notlimited to, total IgG, anti-therapeutic antibody response,agglutination, etc.

In various embodiments, non-human animals as described herein are usedto measure the therapeutic effect of blocking or modulating Lag-3signaling and the effect on gene expression as a result of cellularchanges. In various embodiments, a non-human animal as described hereinor cells isolated therefrom are exposed to a candidate therapeutic thatbinds a humanized Lag-3 polypeptide (or a human portion of a Lag-3polypeptide) on the surface of a cell of the non-human animal and, aftera subsequent period of time, analyzed for effects on Lag-3-dependentprocesses, for example, adhesion, apoptosis, cytokine production,inflammation, proliferation, self-tolerance and viral infection (orresponses).

Non-human animals as described herein express humanized Lag-3polypeptide, thus cells, cell lines, and cell cultures can be generatedto serve as a source of humanized Lag-3 for use in binding andfunctional assays, e.g., to assay for binding or function of a Lag-3antagonist or agonist, particularly where the antagonist or agonist isspecific for a human Lag-3 sequence or epitope or, alternatively,specific for a human Lag-3 sequence or epitope that associates with MHCclass II molecules. In various embodiments, Lag-3 epitopes bound bycandidate therapeutic antibodies can be determined using cells isolatedfrom non-human animals as described herein. In various embodiments, ahumanized Lag-3 polypeptide expressed by a non-human animal as describedherein may comprise a variant amino acid sequence. In variousembodiments, non-human animals as described herein express a humanizedLag-3 polypeptide variant. In various embodiments, the variant ispolymorphic at an amino acid position associated with ligand binding. Invarious embodiments, non-human animals as described herein are used todetermine the effect of ligand binding through interaction with apolymorphic variant of human Lag-3.

Cells from non-human animals as described herein can be isolated andused on an ad hoc basis, or can be maintained in culture for manygenerations. In various embodiments, cells from a non-human animal asdescribed herein are immortalized (e.g., via use of a virus) andmaintained in culture indefinitely (e.g., in serial cultures).

In various embodiments, cells and/or non-human animals as describedherein are used in various immunization regimens to determine theLag-3-mediated functions in the immune response to an antigen. In someembodiments, candidate therapeutics that bind, or block one or morefunctions of, human (or humanized) Lag-3 are characterized in anon-human animal as described herein. Suitable measurements includevarious cellular assays, proliferation assays, serum immunoglobulinanalysis (e.g., antibody titer), cytotoxicity assays andimmunoprecipitation assays (e.g., characterization of ligand-receptorinteractions). In some embodiments, non-human animals as describedherein are used to characterize the Lag-3-mediated functions regulatingan immune response to an antigen. In some embodiments, the antigen isassociated with an autoimmune disease, disorder or condition. In someembodiments, the antigen is associated with an inflammatory disease,disorder or condition. In some embodiments, the antigen is associatedwith cancer or a neoplasm (e.g., HPV, HCV, HIV, EBV, HHV-8, HTLV-1, MCV,etc.). In some embodiments, the antigen is a test antigen (e.g.,ovalbumin or OVA). In some embodiments, the antigen is a targetassociated with a disease or condition suffered by one or more humanpatients in need of treatment.

In various embodiments, non-human animals as described herein are usedin serum assays for determining titers of autoantibody production fortesting the pharmaco-toxicological aspects of candidate therapeuticsthat target human Lag-3. In some embodiments, autoantibody production innon-human animals as described herein results from one or moreautoimmune diseases, disorders or conditions induced in the non-humananimal.

In various embodiments, non-human animals as described herein are usedfor challenge with one or more antigens to determine the therapeuticpotential of compounds or biological agents to modulate Lag-3-dependentregulation of an immune response, including but not limited to thespecific T cell-dependent responses to a given antigen.

In various embodiments, cells and/or non-human animals as describedherein are used in a survival and/or proliferation assay (e.g.,employing T and/or B cells) to screen and develop candidate therapeuticsthat modulate human Lag-3 signaling Activation or loss of Lag-3 plays animportant role in the regulation of T cell responses, and regulation ofself-tolerance by Lag-3 may result from the activation of specificepitopes of the extracellular domain of Lag-3, therefore, candidateLag-3 modulators (e.g., antagonists or agonists) may be identified,characterized and developed using cells of non-human animals asdescribed herein and/or a non-human animal as described herein. In someembodiments, cells and/or non-human animals as described herein are usedin survival or death assay(s) to determine the effect on proliferationor apoptosis of a specific cell(s) (e.g., cancer cells) in the presenceand absence of Lag-3.

In various embodiments, cells and/or non-human animals as describedherein are used in xenotransplantation of heterologous (e.g., human)cells or tissue to determine the Lag-3-mediated functions in thephysiological (e.g., immune) response to the transplanted human cells ortissue. In some embodiments, candidate therapeutics that bind, or blockone or more functions of, human LAG-3 are characterized in a non-humananimal as described herein. Suitable measurements include variouscellular assays, proliferation assays, serum immunoglobulin analysis(e.g., antibody titer), cytotoxicity assays, and characterization ofligand-receptor interactions (immunoprecipitation assays). In someembodiments, non-human animals as described herein are used tocharacterize the Lag-3-mediated functions regulating an immune responseto an antigen. In some embodiments, the antigen is associated with aneoplasm. In some embodiments, the antigen is associated with anautoimmune disease, disorder or condition. In some embodiments, theantigen is associated with an inflammatory disease, disorder orcondition. In some embodiments, the antigen is associated with cancer ora neoplasm. In some embodiments, the antigen is a target associated witha disease or condition suffered by one or more human patients in need oftreatment.

In various embodiments, non-human animals as described herein are usedin transplantation or adoptive transfer experiments to determine thetherapeutic potential of compounds or biological agents to modulateLag-3-dependent regulation of new lymphocytes and their immune function.In various embodiments, non-human animals as described herein aretransplanted with human T cells; in some embodiments, naïve T cells; insome embodiments, activated T cells; in some embodiments, regulatory Tcells (Tregs); in some embodiments, memory T cells.

In various embodiments, cells of non-human animals as described hereinare used in T cell assays to determine the therapeutic potential ofcompounds or biological agents to modulate Lag-3-dependent regulation ofT cell-dependent response and function. Exemplary T cell assays include,but are not limited to, ELISpot, intracellular cytokine staining, majorhistocompatibility complex (MHC) restriction, viral suppression assays,cytotoxicity assays, proliferation assays and regulatory T cellsuppression assays.

In various embodiments, cells of non-human animals as described hereinare used in cell transmigration assays to screen and develop candidatetherapeutics that modulate human Lag-3. Cell transmigration involves themigration of cells across the endothelium and transmigration assayspermit the measurement of interactions with, and transmigration of, theendothelium by leukocytes or tumor cells.

In various embodiments, cells of non-human animals as described hereinare used in tumor cell growth (or proliferation) assays to determine thetherapeutic potential of compounds or biological agents to modulateLag-3-dependent regulation, apoptosis, and/or inhibition of tumor cells.

In various embodiments, cells of non-human animals as described hereinare used in cytokine production assays to determine the therapeuticpotential of compounds or biological agents to modulate Lag-3-dependentregulation of cytokine release from T cells. In some embodiments, cellsof non-human animals as described herein are used for detection (and/ormeasurement) of intracellular cytokine release resulting frominteraction of humanized Lag-3 with a drug targeting human LAG-3 or aLag-3 binding partner (e.g., MHC class II).

In various embodiments, an autoimmune disease, disorder or condition isinduced in one or more non-human animals as described herein to providean in vivo system for determining the therapeutic potential of compoundsor biological agents to modulate Lag-3-dependent regulation of one ormore functions of the autoimmune disease, disorder or condition.Autoimmune diseases, disorders or conditions may be induced in one ormore non-human animals as described herein followed by administration ofone or more compounds or biological agents as desired.

Non-human animals as described herein provide an in vivo system for theanalysis and testing of a drug or vaccine. In various embodiments, acandidate drug or vaccine may be delivered to one or more non-humananimals as described herein, followed by monitoring of the non-humananimals to determine one or more of the immune response to the drug orvaccine, the safety profile of the drug or vaccine, or the effect on adisease or condition. In some embodiments, the vaccine targets a virussuch as, for example, human immunodeficiency virus (HIV) or hepatitisvirus (e.g., HCV). Exemplary methods used to determine the safetyprofile include measurements of toxicity, optimal dose concentration,efficacy of the drug or vaccine, and possible risk factors. Such drugsor vaccines may be improved and/or developed in such non-human animals.

Non-human animals as described herein provide an in vivo system forassessing the pharmacokinetic properties of a drug targeting humanLAG-3. In various embodiments, a drug targeting human LAG-3 may bedelivered or administered to one or more non-human animals as describedherein, followed by monitoring of, or performing one or more assays on,the non-human animals (or cells isolated therefrom) to determine theeffect of the drug on the non-human animal. Pharmacokinetic propertiesinclude, but are not limited to, how an animal processes the drug intovarious metabolites (or detection of the presence or absence of one ormore drug metabolites, including, toxic metabolites), drug half-life,circulating levels of drug after administration (e.g., serumconcentration of drug), anti-drug response (e.g., anti-drug antibodies),drug absorption and distribution, route of administration, routes ofexcretion and/or clearance of the drug. In some embodiments,pharmacokinetic and pharmacodynamic properties of drugs (e.g., PD-1modulators) are monitored in or through the use of non-human animals asdescribed herein.

In some embodiments, performing an assay includes determining the effecton the phenotype and/or genotype of the non-human animal to which thedrug is administered. In some embodiments, performing an assay includesdetermining lot-to-lot variability for a Lag-3 modulator (e.g., anantagonist or an agonist). In some embodiments, performing an assayincludes determining the differences between the effects of a drugtargeting Lag-3 administered to a non-human animal as described hereinand a reference non-human animal. In various embodiments, referencenon-human animals may have a modification as described herein, amodification that is different from one as described herein (e.g., onethat has a disruption, deletion or otherwise non-functional Lag-3 geneand/or Pdcd1 gene, or humanization of a Pdcd1 gene) or no modification(i.e., a wild-type non-human animal).

Exemplary parameters that may be measured in non-human animals (or inand/or using cells isolated therefrom) for assessing the pharmacokineticproperties of a drug targeting human LAG-3 include, but are not limitedto, agglutination, autophagy, cell division, cell death,complement-mediated hemolysis, DNA integrity, drug-specific antibodytiter, drug metabolism, gene expression arrays, metabolic activity,mitochondrial activity, oxidative stress, phagocytosis, proteinbiosynthesis, protein degradation, protein secretion, stress response,target tissue drug concentration, non-target tissue drug concentration,transcriptional activity and the like. In various embodiments, non-humananimals as described herein are used to determine a pharmaceuticallyeffective dose of a Lag-3 modulator.

Non-human animals as described herein provide an improved in vivo systemfor the development and characterization of candidate therapeutics foruse in cancer. In various embodiments, non-human animals as describedherein may be implanted with a tumor, followed by administration of oneor more candidate therapeutics. In some embodiments, candidatetherapeutics may include a multi-specific antibody (e.g., a bi-specificantibody) or an antibody cocktail; in some embodiments, candidatetherapeutics include combination therapy such as, for example,administration of mono-specific antibodies dosed sequentially orsimultaneously. The tumor may be allowed sufficient time to beestablished in one or more locations within the non-human animal. Tumorcell proliferation, growth, survival, etc. may be measured both beforeand after administration with the candidate therapeutic(s). Cytoxicityof candidate therapeutics may also be measured in the non-human animalas desired.

Non-human animals as described herein may be used to develop one or moredisease models to evaluate or assess candidate therapeutics and/ortherapeutic regimens (e.g., monotherapy, combination therapy, dose rangetesting, etc.) to effectively treat diseases, disorders or conditionsthat affect humans. Various disease conditions may be established innon-human animals as described herein followed by administration of oneor more candidate molecules (e.g., drugs targeting Lag-3) so thatefficacy of the one or more candidate molecules in a disease conditioncan determined. In some embodiments, disease models include autoimmune,inflammatory and/or neoplastic diseases, disorders or conditions.

To give but one example, non-human animals as described herein providean improved animal model for prophylactic and/or therapeutic treatmentof a tumor or tumor cells. In various embodiments, non-human animals asdescribed herein may be implanted with one or more tumor cells, followedby administration of one or more candidate therapeutics (e.g.,antibodies). In some embodiments, administration of one or morecandidate therapeutics is performed subsequent to (e.g., minutes orhours but typically on the same day as) implantation of one or moretumor cells and one or more candidate therapeutics are evaluated innon-human animals as described herein for efficacy in preventingestablishment of a solid tumor and/or growth of tumor cells in saidnon-human animals. In some embodiments, administration of one or morecandidate therapeutics is performed subsequent to (e.g., days after)implantation of one or more tumor cells and, in some certainembodiments, after a sufficient time such that one or more implantedtumor cells have reached a predetermined size (e.g., volume) innon-human animals as described herein; and one or more candidatetherapeutics are evaluated for efficacy in treatment of one or moreestablished tumors. Non-human animals may be placed into differenttreatment groups according to dose so that an optimal dose or dose rangethat correlates with effective treatment of an established tumor can bedetermined.

Non-human animals as described herein provide an in vivo system for thedevelopment and/or characterization of combination therapies includingdrugs targeting Lag-3 and PD-1 for use in cancer. In variousembodiments, non-human animals as described herein may be implanted withone or more tumor cells, followed by administration of drugs (e.g.,antibodies) targeting human LAG-3 and human PD-1. In some embodiments,administration of drugs targeting human Lag-3 and human PD-1 isperformed subsequent to (e.g., minutes, hours or days after)implantation of one or more tumor cells and combination therapy isevaluated in non-human animals as described herein for efficacy inpreventing establishment of a solid tumor and/or growth of tumor cellsin said non-human animals. In some embodiments, administration of drugstargeting human LAG-3 and human PD-1 is performed after a sufficienttime such that one or more implanted tumor cells have reached apredetermined size (e.g., volume) in non-human animals as describedherein; and combination therapy is evaluated for efficacy in treatmentof one or more established tumors. Non-human animals may be placed intodifferent treatment groups (including monotherapy groups) so that anoptimal treatment regimen that correlates with effective treatment of anestablished tumor can be determined.

Various disease conditions may be established in non-human animals asdescribed herein followed by administration of one or more candidatemolecules (e.g., drugs targeting Lag-3) so that efficacy of the one ormore candidate molecules in a disease condition can determined. In someembodiments, disease models include autoimmune, inflammatory and/orneoplastic diseases, disorders or conditions.

Candidate molecules can be administered to non-human animal diseasemodels using any method of administration including parenteral andnon-parenteral routes of administration. Parenteral routes include,e.g., intravenous, intraarterial, intraportal, intramuscular,subcutaneous, intraperitoneal, intraspinal, intrathecal,intracerebroventricular, intracranial, intrapleural or other routes ofinjection. Non-parenteral routes include, e.g., oral, nasal,transdermal, pulmonary, rectal, buccal, vaginal, ocular. Administrationmay also be by continuous infusion, local administration, sustainedrelease from implants (gels, membranes or the like), and/or intravenousinjection. When a combination therapy is evaluated in non-human animalsas described herein, candidate molecules can be administered via thesame administration route or via different administration routes. When adosing regimen is evaluated in non-human animals as described herein,candidate molecules may be administered at bimonthly, monthly,triweekly, biweekly, weekly, daily, at variable intervals and/or inescalating concentrations to determine a dosing regimen thatdemonstrates a desired therapeutic or prophylactic effect in a non-humananimal in which one or more disease models has been established.

Non-human animals as described herein provide an improved in vivo systemfor the development and characterization of candidate therapeutics foruse in infectious diseases. In various embodiments, non-human animals asdescribed herein may be infected by injection with a virus (e.g., MHV,HIV, HCV, EBV, etc.) or pathogen (e.g., bacteria), followed byadministration of one or more candidate therapeutics. In someembodiments, candidate therapeutics may include a multi-specificantibody (e.g., a bi-specific antibody) or an antibody cocktail; in someembodiments, candidate therapeutics include combination therapy such as,for example, administration of mono-specific antibodies dosedsequentially or simultaneously; in some embodiments, candidatetherapeutics may include a vaccine. The virus or pathogen may be allowedsufficient time to be established in one or more locations or cellswithin the non-human animal so that one or more symptoms associated withinfection of the virus or pathogen develop in the non-human animal. Tcell proliferation and growth may be measured both before and afteradministration with the candidate therapeutic(s). Further, survival,serum and/or intracellular cytokine analysis, liver and/or spleenhistopathology may be measured in non-human animals infected with thevirus or pathogen. In some embodiments, non-human animals as describedherein are used to determine the extent of organ damage associated withviral infection. In some embodiments, non-human animals as describedherein are used to determine the cytokine expression profile in variousorgans of non-human animals infected with a particular virus.

Non-human animals as described herein can be employed to assess theefficacy of a therapeutic drug targeting human cells. In variousembodiments, a non-human animal as described herein is transplanted withhuman cells, and a drug candidate targeting such human cells isadministered to such non-human animal. The therapeutic efficacy of thedrug is then determined by monitoring the human cells in the non-humananimal after the administration of the drug. Drugs that can be tested inthe non-human animals include both small molecule compounds, i.e.,compounds of molecular weights of less than 1500 kD, 1200 kD, 1000 kD,or 800 daltons, and large molecular compounds (such as proteins, e.g.,antibodies), which have intended therapeutic effects for the treatmentof human diseases and conditions by targeting (e.g., binding to and/oracting on) human cells.

In some embodiments, the drug is an anti-cancer drug, and the humancells are cancer cells, which can be cells of a primary cancer or cellsof cell lines established from a primary cancer. In these embodiments, anon-human animal as described herein is transplanted with human cancercells, and an anti-cancer drug is given to the non-human animal. Theefficacy of the drug can be determined by assessing whether growth ormetastasis of the human cancer cells in the non-human animal isinhibited as a result of the administration of the drug.

In specific embodiments, the anti-cancer drug is an antibody molecule,which binds an antigen on human cancer cells. In particular embodiments,the anti-cancer drug is a bi-specific antibody that binds to an antigenon human cancer cells, and to an antigen on other human cells, forexample, cells of the human immune system (or “human immune cells”) suchas T and B cells.

Kits

The present invention further provides a pack or kit comprising one ormore containers filled with at least one non-human animal, non-humancell, DNA fragment (or construct), and/or targeting vector as describedherein. Kits may be used in any applicable method (e.g., a researchmethod). Optionally associated with such container(s) can be a notice inthe form prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, which noticereflects (a) approval by the agency of manufacture, use or sale forhuman administration, (b) directions for use, or both, or a contractthat governs the transfer of materials and/or biological products (e.g.,a non-human animal or non-human cell as described herein) between two ormore entities.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments, which are given forillustration and are not intended to be limiting thereof.

EXAMPLES

The following examples are provided so as to describe to those ofordinary skill in the art how to make and use methods and compositionsof the invention, and are not intended to limit the scope of what theinventors regard as their invention. Unless indicated otherwise,temperature is indicated in Celsius, and pressure is at or nearatmospheric.

Example 1. Humanization of an Endogenous Lymphocyte-Activation Gene 3

This example illustrates exemplary methods of humanizing an endogenousLymphocyte-activation gene 3 (Lag-3) in a non-human mammal such as arodent (e.g., a mouse). The methods described in this example can beemployed to humanize an endogenous Lag-3 gene of a non-human animalusing any human sequence, or combination of human sequences (or sequencefragments) as desired. In this example, a 1,741 bp synthetic DNAfragment containing exons 2, 3 and 4 of a human LAG3 gene that appearsin GenBank accession NM_002286.5 (SEQ ID NO:9) is employed forhumanizing an endogenous Lag-3 gene of a mouse. Alignment of mouse,human, and exemplary humanized protein generated herein, with humanizedregion underlined, is depicted in FIG. 2. FIG. 3 shows a targetingvector for humanization of the genetic material encoding amino acids21-260 of a rodent Lag-3 polypeptide that was constructed usingVELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 andValenzuela et al., 2003, Nature Biotech. 21(6):652-659; hereinincorporated by reference).

Briefly, mouse bacterial artificial chromosome (BAC) clone bMQ-400O17(Invitrogen) was modified to delete the sequence containing exons 2-4(1,750 bp) of an endogenous Lag-3 gene and insert exons 2, 3 and 4 of ahuman LAG-3 gene using a 1,741 bp synthetic DNA fragment, which encodesamino acids 21-260 of a human LAG-3 polypeptide. Endogenous DNAcontaining exons 1, 5, 6, 7 and 8 as well as the 5′ and 3′ untranslatedregions (UTRs) were retained. Sequence analysis of the 1,741 bpsynthetic DNA fragment (i.e., corresponding to exons 2-4 of a human LAG3gene) confirmed all human LAG-3 exons and splicing signals. Sequenceanalysis revealed that the sequence matched the reference genome andLAG-3 transcript NM_002286.5.

The 1,741 bp synthetic DNA fragment was synthesized by Genescript Inc.(Piscataway, N.J.) and cloned into an ampicillin-resistant plasmidvector. Unique restriction enzyme recognition sites were employed toligate a ˜4,996 bp self-deleting neomycin cassette flanked byrecombinase recognition sites (loxP-hUbl-em7-Neo-pA-mPrm1-Crei-loxP; seeU.S. Pat. Nos. 8,697,851, 8,518,392 and 8,354,389, all of which areincorporated herein by reference). Subsequent selection employedneomycin. The targeting vector was linearized prior to homologousrecombination with mouse BAC clone bMQ-400O17. By design, the junctionbetween the human LAG-3 1,741 bp fragment and the mouse downstream 509bp included a portion of intron 4 of a human LAG-3 gene (FIG. 3). Theresulting targeting vector contained, from 5′ to 3′, a 5′ homology armcontaining ˜46 kb of mouse genomic DNA from BAC clone bMQ-400O17, 1,741bp synthetic DNA fragment (corresponding to exons 2-4 and a portion ofintron 4 of a human LAG-3 gene), a self-deleting neomycin cassetteflanked by loxP sites, and ˜53 kb of mouse genomic DNA from BAC clonebMQ-400O17.

The modified bMQ-400O17 BAC clone described above was used toelectroporate mouse embryonic stem (ES) cells to create modified EScells comprising an endogenous Lag-3 gene that is humanized from exon 2through exon 4 including a portion of intron 4 (i.e., deletion of 1,750bp of an endogenous Lag-3 gene and insertion of 1,741 bp of human LAG-3sequence). Positively targeted ES cells containing a humanized Lag-3gene were identified by an assay (Valenzuela et al., supra) thatdetected the presence of the human LAG-3 sequences (e.g., exons 2-4) andconfirmed the loss and/or retention of mouse Lag-3 sequences (e.g.,exons 2-4 and/or exons 1, 4, 5, 6, 7 and 8). Table 1 sets forth theprimers and probes that were used to confirm humanization of anendogenous Lag-3 gene as described above (FIG. 4).

The nucleotide sequence across the upstream insertion point included thefollowing, which indicates endogenous mouse sequence (contained withinthe parentheses below) contiguous with human LAG-3 genomic sequence atthe insertion point:

(SEQ ID NO: 12) (CATGATGTTT CTTTCTTAGG AAAGCCAGGG CATTTCTCTATTCTCCAATC TCTTGGCTCA ATGCCCTTGG CCTCTCTTTTGTTCCACTAG) TGAAGCCTCT CCAGCCAGGG GCTGAGGTCCCGGTGGTG TGGGCCCAG GAGGGGGCTC CTGCCCAGCT CCC (FIG. 9A).

The nucleotide sequence across the 5′ end of the self-deleting neomycincassette included the following, which indicates human LAG-3 genomicsequence contiguous with cassette sequence (contained within theparentheses below with a SalI-XhoI compatible end italicized and a loxPsequence in bold font) downstream of the insertion point:

(SEQ ID NO: 13) TTCACATTTG ACCACAACTC CTTCCTGCCC CCCTTGTCACCTCCCCTAAC (GTCGAG ATAACTTCG TATAATGTAT GCTATACGAAGTTAT ATGCATGGCC TCCGCGCCGG GTTTTGGCGC CTCCCGCGGGCGCCCCCCTC CTCACGGCGA GCGCTGCCAC GTCAGACGAAGGGCGCAGCG AGCGTCCTGA) (FIG. 9B).

The nucleotide sequence across the downstream insertion point at the 3′end of the self-deleting neomycin cassette included the following, whichindicates cassette sequence (contained within the parentheses below witha loxP site in bold, an I-CeuI recognition site underlined and an NheIrecognition site italicized) contiguous with mouse Lag-3 genomicsequence:

(SEQ ID NO: 14) (TTTCACTGCAT TCTAGTTGTG GTTTGTCCAA ACTCATCAATGTATCTTATC ATGTCTGGA ATAACTTCGT ATAATGTATGCTATACGAAG TTAT GCTAGTAACT ATAACGGTCC TAAGGTAGCG AGCTAGC) GACCCCCAAA ACTTTCTCAG CTGCGTGTGGTCTCACTCCA CATCACTTTG TTTCAGTGTC CAAACCATTTTCTCTCTGGG CATCTTTTAG (FIG. 9C).

The nucleotide sequence across the upstream insertion point afterdeletion of the neomycin cassette (77 bp remaining in intron 4) includedthe following, which indicates mouse and human genomic sequencejuxtaposed with remaining cassette sequence loxP sequence (containedwithin the parentheses below with a SalI-XhoI compatible end italicized,a loxP site in bold, an I-CeuI restriction site underlined and an NheIrestriction site italicized):

(SEQ ID NO: 15) ACGTCTCCAT CATGTATAAC CTCACTGTTC TGGGTAACTCCCCCACTCT GCTTCACATT TGACCACAAC TCCTTCCTGCCCCCCTTGTC ACCTCCCCT AAC (GTCGAG ATAACTTCGTATAATGTATGC TATACGAAGT TAT GCTAGTA ACTATAACGGTCCTAAGGTA GCGA GCTAGC) GACCCCCAAA ACTTTCTCAGCTGCGTGTGG TCTCACTCCA CATCACTTTG TTTCAGTGTCCAAACCATTT TCTCTCTGG GCATCTTTTA GCTGCTGTCTC (FIG. 9D).

Positive ES cell clones were then used to implant female mice using theVELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirouet al., 2007, Nature Biotech. 25(1):91-99) to generate a litter of pupscontaining an insertion of human LAG-3 exons 2-4 and part of human LAG-3intron 4 into an endogenous Lag-3 gene of a mouse. Mice bearing thehumanization of exons 2-4 (i.e., the 1,741 bp synthetic DNA fragment) ofan endogenous Lag-3 gene were again confirmed and identified bygenotyping of DNA isolated from tail snips using an assay as previouslydescribed (Valenzuela et al., supra) that detected the presence of thehuman LAG-3 gene sequences (FIG. 4). Pups are genotyped and cohorts ofanimals heterozygous for the humanized Lag-3 gene construct are selectedfor characterization.

TABLE 1 Name Primer/Probe Sequence (5′-3′) 7184 ForwardGAGGCTGCTGACGGTCAAG (SEQ ID NO: 16) mTU ProbeTTAGGCAGGTTAACTTTATCCTCAAAGCA (SEQ ID NO: 17) ReverseGCCACGAAGAAGATGCACTCAAG (SEQ ID NO: 18) 7184 ForwardGTCCCGGGTCTCTTGGAGAT (SEQ ID NO: 19) mTD ProbeCCACCCATAAACATCCCCAGGTTTCA (SEQ ID NO: 20) Reverse CCGCTCATTCCAAGTCAGTTC(SEQ ID NO: 21) 7184 hTU Forward CGGTTGGTGGTCAAGAGAAC (SEQ ID NO: 22)Probe CGGGCTTTCTCATCCTCAACGGG (SEQ ID NO: 23) ReverseGGCGGGAAAGAGAATGGAGTTG (SEQ ID NO: 24) 7184 hTD ForwardAGCCCTGCTGTGTTGGGAAA (SEQ ID NO: 25) Probe TGTTTCCAGTGGGCTGATGAAGTC(SEQ ID NO: 26) Reverse TGGCAGTCACTGTGCAAG (SEQ ID NO: 27)

Example 2. Expression of Humanized Lag-3 on Activated T Cells

This Example demonstrates that non-human animals (e.g., rodents)modified to contain a humanized Lag-3 gene according to Example 1express a humanized Lag-3 polypeptide on the surface of activatedlymphocytes. In particular, activated T cells from wild type mice andmice homozygous for humanized Lag-3 (as described above) were stainedwith anti-Lag-3 antibodies to determine the expression of LAG-3 in Tcells stimulated with anti-CD3/anti-CD28 antibodies. Further, expressionof PD-1 and LAG-3 was examined on T cells of humanized Lag-3 mice thatfurther contained a humanized Pdcd1 (PD-1) gene (double humanizedLag3xPD1 mice).

First, double humanized Lag3xPD1 mouse embryonic stem (ES) cells weregenerated using the Lag-3 targeting vector described above (seeExample 1) in humanized Pdcd1 mouse ES cells (for humanized PD-1 miceand embryonic stems cells see U.S. patent application Ser. No.14/744,592, filed 19 Jun. 2015 and published as US 2015-0366174 A1(including particularly Example 1 therein), and International PatentApplication No. PCT/US15036649, filed 19 Jun. 2015 and published asWO2015196051; both of which are incorporated herein by reference).Double humanized mouse ES cells were created by electroporation of theLag-3 targeting vector as described in Example 1 into C57BL/6N mouseembryonic stem cells that contained a humanized Pdcd1 gene that encodeda PD-1 polypeptide having a human portion and a mouse portion, whichhuman portion included the extracellular domain of a human PD-1polypeptide. Double humanized ES cell clones were then used to implantfemale mice using the VELOCIMOUSE® method (see, e.g., U.S. Pat. No.7,294,754 and Poueymirou et al., 2007, Nature Biotech. 25(1):91-99) togenerate a litter of pups containing both humanized genes (i.e., Lag-3and Pdcd1). The mouse colony was expanded by interbreeding.

Briefly, spleens were harvested and processed from wild type, humanizedLag-3 mice made according to Example 1, humanized PD-1 mice, and doublehumanized Lag3xPD1 mice (described above) into single cell suspensionsby mechanical dissociation. Cells were washed in media (RPMIsupplemented with 10% FBS), re-suspended at 1×10⁶/mL and 200 μL (200,000cells) were plated in 96-well plates. Cells in selected wells werestimulated with anti-CD3 and anti-CD28 antibodies (both at 1 μg/mL) for72 hours. Cells were stained for FACS according to manufacturer'sspecifications with antibodies recognizing CD4 and CD8, BV421 (BrilliantViolet 421™-conjugated antibody to human PD-1 (clone EH12.2H7,Biolegend), BV421-conjugated antibody to mouse PD-1 (clone J43, BDBiosciences), allophycocyanin-conjugated antibody to human LAG-3 (clone3DS223H, eBioscience), allophycocyanin-conjugated antibody to mouseLag-3 (clone C9B7W, Biolegend) or corresponding isotype controls.Stained cells were run on LSRII flow cytometer and data was analyzedusing Flowjo software. CD4⁺ T cells were gated (CD19⁻CD4⁺) forexpression of human and mouse Lag-3. Representative results are shown inFIG. 5.

As shown in FIG. 5, activated CD4⁺ T cells from wild type mouse (FIG. 5,left column) show robust expression of mouse PD-1 (bottom left) andmouse Lag-3 (2nd from top left), but a complete lack of human PD-1 andhuman LAG-3 expression. This demonstrated that anti-human PD-1 andanti-human LAG-3 antibodies do not cross-react with mouse PD-1 and mouseLag-3 proteins, respectively. Activated CD4⁺ T cells from singlehumanized Lag-3 mice detectably express humanized Lag-3 and mouse PD-1,but not mouse Lag-3 and human PD-1 (FIG. 5, second column from left).Correspondingly, activated CD4⁺ T cells from single humanized Pdcd1 miceexpress humanized PD-1 and mouse Lag-3, but lack any expression of mousePD-1 and human LAG-3 (FIG. 5, third column from left). Double humanizedLag3xPD1 mice demonstrated expression of human but not mouse PD-1 andLag-3 proteins, which confirmed that full-length mouse PD-1 and Lag-3polypeptides are not produced in these mice.

Taken together, this Example demonstrates that mice bearing a humanizedLag-3 gene as described in Example 1 express a Lag-3 polypeptide thatcomprises a human portion and an endogenous mouse portion, which humanportion is detectably expressed via recognition by an antibody thatrecognizes human LAG-3 polypeptide. This Example also demonstrates thatdouble humanized Lag3xPD1 mice expressing both humanized PD-1 andhumanized Lag-3 polypeptides provide an in vivo system for testing theefficacy of anti-Lag-3 and/or anti-PD1 antibodies.

Example 3. In Vivo Efficacy of LAG3 and PD1 Modulators

This Example demonstrates that non-human animals (e.g., rodents)modified to contain a humanized Lag-3 gene according to Example 1 can beused in an in vivo assay to screen both Lag-3 and PD-1 modulators (e.g.,anti-Lag-3 and anti-PD-1 antibodies) and determine variouscharacteristics such as, for example, inhibition of tumor growth and/orkilling of tumor cells. In this Example, anti-Lag-3 and anti-PD-1antibodies are screened in mice homozygous for humanization of anendogenous Lag-3 gene as described in Example 1 and homozygous forhumanization of an endogenous Pdcd1 gene to determine the efficacy ofmono- and combination therapy using anti-Lag-3 and anti-PD1 antibodies.

MC38, a mouse adenocarcinoma cell line, originated from a C57BL/6 mousestrain, was obtained from the National Health Institute depository. TheMC38.Ova cell line was engineered by stable lenti-viral transduction toexpress transmembrane chicken ovalbumin antigen (Ova) to increase tumorimmunogenicity. Cell lines were tested for human and rodent pathogensbefore being implanted into mice. Cells were suspended in 100 μlserum-free RPMI medium and implanted subcutaneously into the mouseflank.

Briefly, in vivo efficacy of anti-Lag-3 and anti-PD-1 antibodies aloneand in combination was examined using MC38.Ova tumors implanted indouble humanized Lag3xPD1 mice. Mice were implanted subcutaneously withMC38.Ova cells on day 0 and randomized into four treatment groups(n=7-12 for each treatment group). Mice were administered a controlantibody (n=7, 25 mg/kg), anti-Lag-3 (n=12, 25 mg/kg), anti-PD-1 (n=12,10 mg/kg), or anti-Lag-3 and anti-PD-1 antibody combination (n=12, 25mg/kg and 10 mg/kg, respectively) via intraperitoneal injection on days3, 7, 10, 14 and 17. Tumor volumes were monitored by caliper measurementtwice per week for the duration of the experiment (32 days) and tumorfree animals were monitored for the absence of tumor recurrence for upto 80 days. Representative results are shown in FIG. 6.

As shown in FIG. 6, anti-PD-1 monotherapy resulted in tumor growthinhibition, with tumor regression in 2 out of 12 (17%) animals whereasanti-Lag-3 monotherapy was not significantly efficacious in thisexperiment. Tumor regression was observed in 1 out of 12 mice treatedwith anti-Lag-3 antibody. There were no tumor-free mice on day 32 forthe control group. By contrast, combination therapy with anti-Lag-3 andanti-PD-1 demonstrated robust inhibition of MC38.Ova tumor growth,resulting in 5 out of 12 (42%) tumor-free mice by the end of experiment(FIG. 6). None of the tumor-free mice showed tumor recurrence for 80days post-implantation, which indicated long-lasting effects ofcombination immunotherapy.

In a separate experiment, double humanized Lag3xPD1 mice were inoculatedwith MC38.Ova tumor cells subcutaneously on day 0. On day 10 mice withan average tumor volume of 100 mm³ were selected and randomized intofour treatment groups. Mice were administered anti-Lag-3 antibody (n=9,25 mg/kg), anti-PD-1 antibody (n=10, 10 mg/kg), anti-lag-3 antibody andanti-PD-1 antibody combination (N=11, 25 mg/kg and 10 mg/kg,respectively) or a control antibody (n=7, 25 mg/kg) by IP injection ondays 10, 14, 17, 22. Tumor volumes were monitored for 28 days post tumorimplantation.

As shown in FIG. 7, treatment of MC38.ova tumor-bearing humanized mice(established tumors) with a combination of anti-hPD-1 and anti-hLAG-3antibodies triggered activation of intratumoral and peripheral T cells.Combination of anti-hPD-1 and anti-hLAG-3 antibodies demonstrated robustinhibition of MC38.Ova tumor growth. Combination therapy in these micealso resulted in significant increase in animal survival, as well asduration of survival (data not shown).

Taken together, this Example demonstrates that non-human animalsdescribed herein can be used to assess the in vivo efficacy of drugs(e.g., one or more antibodies) targeting Lag-3 and/or PD-1, and suchanimals are useful in discriminating the therapeutic and prophylacticeffect of anti-Lag-3 monotherapy and/or combination therapy usinganti-PD-1 antibodies. Moreover, non-human animals described herein canbe used to assess the extent to which drugs targeting Lag-3 or PD-1 caninhibit tumor growth and/or mediate killing of tumor cells.

EQUIVALENTS

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated by those skilled in the art thatvarious alterations, modifications, and improvements will readily occurto those skilled in the art. Such alterations, modifications, andimprovements are intended to be part of this disclosure, and areintended to be within the spirit and scope of the invention.Accordingly, the foregoing description and drawing are by way of exampleonly and the invention is described in detail by the claims that follow.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, (e.g., in Markush group orsimilar format) it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth in so many wordsherein. It should also be understood that any embodiment or aspect ofthe invention can be explicitly excluded from the claims, regardless ofwhether the specific exclusion is recited in the specification.

Those skilled in the art will appreciate typical standards of deviationor error attributable to values obtained in assays or other processesdescribed herein. The publications, websites and other referencematerials referenced herein to describe the background of the inventionand to provide additional detail regarding its practice are herebyincorporated by reference.

What is claimed is:
 1. A mouse whose genome comprises a humanizedLymphocyte activation gene 3 (Lag-3) gene at an endogenous Lag-3 locus,wherein a humanized Lag-3 polypeptide is expressed from the humanizedLag-3 gene on the surface of activated T-lymphocytes in the mouse,wherein the humanized Lag-3 polypeptide comprises (i) the first twoN-terminal immunoglobulin-like domains of a human LAG-3 polypeptide and(ii) the transmembrane and intracellular domains of an endogenous mouseLag-3 polypeptide, and wherein the humanized Lag-3 gene is operablylinked to the endogenous mouse Lag-3 promoter at the endogenous Lag-3locus.
 2. The mouse of claim 1, wherein the humanized Lag-3 polypeptidecomprises amino acids 29-260 of the human LAG-3 polypeptide, wherein thehuman LAG-3 polypeptide comprises the amino acid sequence as set forthin SEQ ID No.
 6. 3. The mouse of claim 1, wherein the humanized Lag-3gene comprises endogenous mouse Lag-3 exons 1, 5, 6, 7 and
 8. 4. Themouse of claim 1, wherein the humanized Lag-3 gene comprises exons 2-4of a human LAG-3 gene.
 5. A humanized Lag-3 polypeptide produced by themouse of claim
 1. 6. An isolated cell or tissue of the mouse of claim 1,wherein the genome of the isolated cell or tissue comprises thehumanized Lag-3 gene at the endogenous Lag-3 locus.
 7. A method ofmaking a mouse comprising modifying a mouse genome so that the modifiedgenome comprises a humanized Lag-3 gene at an endogenous Lag-3 locus,thereby making the mouse, wherein a humanized Lag-3 polypeptide isexpressed from the humanized Lag-3 gene on the surface of activatedT-lymphocytes in the mouse, and wherein the humanized Lag-3 polypeptidecomprises (i) the first two N-terminal immunoglobulin-like domains of ahuman LAG-3 polypeptide and (ii) the transmembrane and intracellulardomains of an endogenous mouse Lag-3 polypeptide, and wherein thehumanized Lag-3 gene is operably linked to the endogenous mouse Lag-3promoter at the endogenous Lag-3 locus.
 8. The method of claim 7,comprising (a) integrating a genomic fragment into an endogenous Lag-3gene in a mouse ES cell, the genomic fragment comprising a nucleotidesequence that encodes the first two N-terminal immunoglobulin-likedomains of the human LAG-3 polypeptide, thereby obtaining a geneticallymodified mouse embryonic stem cell; and, (c) creating a mouse using thegenetically modified mouse ES cell of (a).
 9. The method of claim 7,wherein the humanized Lag-3 gene comprises exons 2-4 of a human LAG-3gene.
 10. The method of claim 7, wherein the humanized Lag-3 geneencodes at least amino acids 29-260 of the human LAG-3 polypeptide,wherein the human LAG-3 polypeptide comprises the amino acid sequence asset forth in SEQ ID No. 6.